WO2026090322A1 - Compositions and methods for performing spatially resolved transcriptomics - Google Patents

Abstract

Provided herein are compositions and methods for performing micro isoform ST spatial transcriptomics (miST).

Description

COMPOSITIONS AND METHODS FOR PERFORMING SPATIALLY RESOLVED TRANSCRIPTOMICS

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under HG011014 awarded by the National Institutes of Health. The government has certain rights in the invention.

INCORPORATION-B Y-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

Applicant hereby incorporates by reference the Sequence Listing material filed in electronic form herewith. This file is labeled “NYG-LIPP-250PCT.xml” (created October 22, 2025 and 184,013 bytes in size).

BACKGROUND

Nucleic acids are generally measured and analyzed solely based on their sequence. However, much of their biological functions and current status is encoded in other metadata features including their secondary structure, chemical modifications, etc.

Current spatially resolved sequencing methods such as Decoder-seq and MAGIC-seq rely on signal amplification strategies (e.g., polymerase chain reaction) to append barcode sequences and spatial information onto nucleic acids, thus losing the chemical metadata stored in the native molecule.

What is needed is improved compositions and methods for performing spatially resolved, transcriptome-wide measurements of RNA and chemical modifications.

SUMMARY OF THE INVENTION

In one aspect, provided herein is a solid substrate for performing spatially resolved transcriptome sequencing of one or more biological specimens, the solid substrate having a surface comprising: an array of capture regions comprising substrate oligonucleotides bound to the solid substrate, the substrate oligonucleotides comprising a cleavable motif, a UMI, a first sector barcode, a second sector barcode, a first pixel barcode, a second pixel barcode, and a capture sequence, each capture region of the array being identifiable by the substrate oligonucleotide bound thereto; wherein the first sector barcode is common to the substrate oligonucleotides of a series of adjacent rows of capture regions of the array, and the second sector barcode is a common to the substrate oligonucleotides of a series of adjacent columns of capture regions of the array, each combination of first sector barcode and second sector barcode defining a sector; and each sector comprising multiple capture regions, each capture region comprising substrate oligonucleotides comprising a unique combination of first pixel barcode and second pixel barcode, wherein each unique combination of first pixel barcode and second pixel barcode defines x and y positions, respectively, within that sector.

In another aspect, provided herein is a solid substrate for performing spatially resolved transcriptome sequencing of one or more biological specimens, the solid substrate having a surface comprising: substrate oligonucleotides bound to the solid substrate, the substrate oligonucleotides comprising a cleavable motif, a UMI, a first pixel barcode, a second pixel barcode, a first sector barcode, a second sector barcode, and a capture sequence, each capture region of the array being identifiable by the substrate oligonucleotide bound thereto; wherein the first sector barcode is common to the substrate oligonucleotides of a series of adjacent rows of capture regions of the array, and the second sector barcode is a common to the substrate oligonucleotides of a series of adjacent columns of capture regions of the array, each combination of first sector barcode and second sector barcode defining a sector; and each sector comprising multiple capture regions, each capture region comprising substrate oligonucleotides comprising a unique combination of first pixel barcode and second pixel barcode, wherein each unique combination of first pixel barcode and second pixel barcode defines x and y positions, respectively, within that sector.

In another aspect, provide herein is a method for generating a solid substrate having a surface comprising an array of capture regions, the capture regions being arranged in rows and columns, the method comprising: i) contacting each row in a series of adjacent rows of a solid substrate with one of a set of first sector barcode oligonucleotides, each first sector barcode oligonucleotide comprising a first sector barcode and a first linker, wherein each first sector barcode oligonucleotide in the set has a different first sector barcode, wherein the first sector barcode oligonucleotides are bound to the solid substrate using a water-soluble amine linker, optionally bis(sulfosuccinimidyl)suberate (BS3), wherein the first sector barcode oligonucleotides comprise a 5’ amine modification: ii) contacting each column in a series of adjacent columns of the solid substrate with one of a set of second sector barcode oligonucleotides, each second sector barcode oligonucleotide comprising a second sector barcode and a second linker, wherein each second sector barcode oligonucleotide in the set has a different second sector barcode, wherein each combination of first sector barcode and second sector barcode defines a sector; iii) contacting each row in the series of adjacent rows of the solid substrate with a set of first pixel barcode oligonucleotides, each first pixel barcode oligonucleotide comprising a first pixel barcode and a third linker, wherein each first pixel barcode oligonucleotide in the set has a different first pixel barcode; iv) contacting each column in the series of adjacent columns of the solid substrate with a set of second pixel barcode oligonucleotides, each second pixel barcode oligonucleotide comprising a second pixel barcode and a fourth linker, wherein each second pixel barcode oligonucleotide in the set has a different second pixel barcode, wherein each combination of first pixel barcode and second pixel barcode defines x and y positions, respectively, within a sector; and v) contacting each column and row of the solid substrate with a capture oligonucleotide comprising a fourth linker and a capture sequence.

In another aspect, provide herein is a method for generating a solid substrate having a surface comprising an array of capture regions, the capture regions being arranged in rows and columns, the method comprising: i) contacting each row in a series of adjacent rows of a solid substrate with one of a set of first sector barcode oligonucleotides, each first sector barcode oligonucleotide comprising a first sector barcode and a first linker, wherein each first sector barcode oligonucleotide in the set has a different first sector barcode, wherein the first sector barcode oligonucleotides are bound to the solid substrate having an amine-reactive functional groups, optionally N-oxysuccinimide, wherein the first sector barcode oligonucleotides comprise a 5’ amine modification: ii) contacting each column in a series of adjacent columns of the solid substrate with one of a set of second sector barcode oligonucleotides, each second sector barcode oligonucleotide comprising a second sector barcode and a second linker, wherein each second sector barcode oligonucleotide in the set has a different second sector barcode, wherein each combination of first sector barcode and second sector barcode defines a sector; iii) contacting each row in the series of adjacent rows of the solid substrate with a set of first pixel barcode oligonucleotides, each first pixel barcode oligonucleotide comprising a first pixel barcode and a third linker, wherein each first pixel barcode oligonucleotide in the set has a different first pixel barcode; iv) contacting each column in the series of adjacent columns of the solid substrate with a set of second pixel barcode oligonucleotides, each second pixel barcode oligonucleotide comprising a second pixel barcode and a fourth linker, wherein each second pixel barcode oligonucleotide in the set has a different second pixel barcode, wherein each combination of first pixel barcode and second pixel barcode defines x and y positions, respectively, within a sector; and v) contacting each column and row of the solid substrate with a capture oligonucleotide comprising a fourth linker and a capture sequence.

In yet another aspect, provided herein is a solid substrate obtained by a method disclosed herein.

In another aspect, provided herein is a method of spatially resolved transcriptome sequencing of one or more biological specimens, the method comprising: a) providing a solid substrate as disclosed herein; b) mounting the one or more biological samples to the solid substrate, the biological samples partially or completely overlaying the array of capture regions; c) performing staining or immunofluorescent labeling of the one or more biological samples; d) capturing one or more images of the one or more biological samples; e) permeabilizing the one or more biological specimens thereby permitting RNA present in the one or more biological specimens to bind capture sequences of the substrate oligonucleotides; f) performing reverse-transcription by addition of a reverse transcriptase under conditions suitable to permit generation of cDNA:RNA hybrid molecules, wherein the reverse transcription includes extension of RNA using the bound substrate oligonucleotide as a template to result in tagging of the RNA with sequences complementary to the first sector barcode, the second sector barcode, the first pixel barcode, and the second pixel barcode of the bound substrate oligonucleotide; and g) sequencing tagged RNA and assigning sequences to a capture region.

Still other aspects and advantages of these compositions and methods are described further in the following detailed description of the preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 A - FIG. 1C show an overview of combinatorial microfluidics devices used for the fabrication of (FIG. 1 A) sixteen 96-by-96 channel subarrays via (FIG. IB) duplex ligation or (FIG. 1C) hybridization-and-extension DNA polymerization.

FIG. 2 shows FISH images (intensities of the white signal indicates poly(d)T presence) of optimizing DNA to reducing agent ratios landing at a 1-1 OpM DNA concentration used in formulations.

FIG. 3 shows FISH images of poly(d)T -stained sector arrays using competing approaches for sector barcode deposition (left) and miST (right).

FIG. 4A and FIG. 4B show FISH stains of miST arrays production for a 16-sector device. (FIG. 4 A) Each individual yellow square represents a single 10pm pixel that has a unique spatial barcode. (FIG. 4B) Zoom in on an array representing one of the sixteen sectors (cyan inset in FIG. 4A). Color code: The poly(dT) capture sequence, deposited across rows with a pixel device, is labeled in yellow. The linker sequence, deposited across columns using a pixel device, is shown in red.

FIG. 5 A and FIG. 5B show immunofluorescence staining of mi ST arrays that were produced using two different formulations. (FIG. 5A) Single-sector array fabricated disuccinimidyl suberate-linked dendrimer surface (left) bis(sulfosuccinimidyl)suberate-linked dendrimer surface (right) and with 10 pm pixels. (FIG.5B) Zoomed image of array insets (white rectangles in FIG. 5A) synthesized using either disuccinimidyl suberate (left) or bis(sulfosuccinimidyl)suberate (right). The poly(dT) capture sequence, deposited across rows with a pixel device, is labeled in yellow. The fiducial sequence, found only in the columns of pixels flanking the array, are labeled in red.

FIG. 6A and FIG. 6B show FISH visualization of the pixel frame probe. (FIG. 6A) A single sector arrays visualized on the glass surface using the complementary frame probe (red) and poly(d)T (yellow). (FIG. 6B) Zoom in (inset from FIG. 6A) on frame probe and showing example image processing for frame probe detection. White squares show the frame probe pixels on the spatial array.

FIG. 7 shows array and tissue registration visualization. A DAPI stained mouse brain tissue on top of a miST spatial array (left) and zoom in (insets from right).

FIG. 8 shows example designs of the first four sector barcodes used in the first PDMS device that attaches oligos to the glass surface. The design indicate the attachment chemistry (5 ’-Amine modification followed by a C6 linker), a stretch of dU nucleotides, a PCR primer handle that is adaptable with multiple sequencing technologies and library preparation strategies (and is fully interchangeable), a degenerate UMI sequence where V and D are degenerate bases to determine the start and stop of the UMI sequence, a sector barcode (where the 4 different sequences exemplify the different sector barcodes) followed by a Linker A sequence which is used in the next PDMS device to hybridize an orthogonal set of sector barcodes through a Linker-A’ sequence.

FIG. 9 shows final barcode design metrics. Each of the pixel barcodes in lOnt long, has 50% GC content, there are no homopolymer repeats, the shared k-mer length was minimized, and the pairwise distance was maximized(tested for Levenshtein and Hamming).

FIG. 10 provides a table showing comparison of sequencing accuracy in combinatorial barcodes synthesized by either hybridization-and-extension (“Klenow”) or ligation. Values shown are the percent of reads which have a flanking DNA adapter sequence detected with either a perfect match to the whitelisted barcode sequences and the percent of reads with fewer than three mismatches (“Hamming Distance < 3”).

FIG. 11 shows immunofluorescence images of spatial gene activity of an adult mouse brain. Intensity of the white signal indicates more or longer mRNA molecules have been spatially transcribed in the cDNA synthesis steps using Decoder-seq cDNA RT conditions (left) and miST conditions (right) on consecutive mouse brain sections. Stronger signal intensities are favorable as they indicate more cDNA product has been transcribed.

FIG. 12 shows immunofluorescence images (intensities of the white signal indicate poly(d)T presence) of optimizing cDNA:mRNA hybrid cleavage reactions from the spatial surface. “Cleavage reaction ON” indicates the cleavage reaction was applied to replicate spatial arrays (columns) while “Cleavage reaction OFF” indicates the conditions where no cleavage enzyme was added to the reaction. Decrease in while signal intensities in the “Cleavage reactions ON” condition” as compared to the “Cleavage OFF” condition indicated successful cleavage of the cDNA:mRNA hybrid.

FIG. 13 shows a workflow for (i) direct RNA capture, (ii) barcoding via reverse transcription, (iii) enzymatic release from slide, (iv) adapter ligation, and (v) nanopore sequencing.

FIG. 14A and FIG. 14B show barcode transfer via DNA-templated extension with reverse transcriptase enzymes. (FIG. 14A) RNA integrity after reverse transcription and first strand cDNA degradation with DNase. The x-axis shows the length of the RNA molecules and the y-axis shows the relative abundance of molecules of a particular length. (FIG. 14B) PCR amplification of a barcoded RNA library using either a full barcode and poly(dT)VN primer or just the barcode sequence. The x-axis shows the length of the cDNA molecules and the y-axis shows the relative abundance of molecules of a particular length.

FIG. 15A - FIG. 15D shows optimized conditions for surface capture and release of full-length RNA-DNA hybrid molecules. Length analysis of DNA/RNA hybrid molecules after surface capture, reverse transcription, and enzymatic release when (FIG. 15 A) no enzyme was included in the reverse transcription step, (FIG. 15B) Maxima H-Minus RT was included using previously demonstrated conditions, (FIG. 15C) the commercially recommended concentration of Ultra Marathon RT was included in the reverse transcription step, or (FIG. 15D) Ultra Marathon RT was used at an optimized concentration in the reverse transcription step. FIG. 16 shows immunofluorescence staining of a mi ST arrays that was produced using a TRIDIA™ surface having amine-reactive N-oxysuccinimide functional groups (SurModics, Inc.)

DETAILED DESCRIPTION OF THE INVENTION

Spatial transcriptomic methods utilize nucleic acid probes immobilized to a solid substrate (e.g., an array) to capture nucleic acids (DNA or RNA) from a biological specimen and subsequently tag the nucleic acids (e.g. with “barcode” sequences) based on their location within the biological specimen (see e.g. WO 2012/140224). The tagged nucleic acids are subsequently analyzed to generate information about the localization, distribution and/or expression of genes in the biological specimen, such as a tissue sample.

While spatial transcriptomics reveal can reveal the histological context of gene expression, existing tools overlook many of the molecular nuances important to understanding that context. Recent advancements in long-read technologies have the potential to add important metadata to RNA-sequencing measurements, including splicing status, isoform identity, poly(A) tail length, and more. However, existing spatial transcriptomics platforms were designed for legacy short-read technologies and integrate poorly with long-read workflows, resulting in shorter-than-expected reads and poor sequencing economy.

Provided herein are compositions and methods for performing micro isoform ST (miST), a spatial long read transcriptomics platform that is compatible with long-read RNA sequencing, including nanopore sequencing. The mi ST platform leverages iterative combinatorial indexing to build each spatial barcode piecewise. This approach requires relatively few reagents for fabrication and can be scaled to build arrays with >1 cm² surface area and >150,000 barcoded spots in a single day. One of the major pain points of commercial platforms is the fraction of reads that cannot be confidently matched to a known spatial barcode, often resulting in loss of more than half of the reads. With miST, the spatial barcodes are designed to be error-robust in nanopore sequencing, resulting in substantial improvement in barcode assignment and overall sequencing economy. Additionally, currently available commercial platforms do not include reverse transcription and library preparation reactions that are optimized for long-read sequencing. We further improved the library preparation to generate longer cDNA amplicons and to require fewer PCR cycles.

Previous technologies for performing spatial transcriptomics include Decoder-seq (Cao J, et al. Decoder-seq enhances mRNA capture efficiency in spatial RNA sequencing. NatBiotechnol. 2024 Nov;42(ll): 1735-174) , MAGIC-seq (Zhu J, et al. Custom microfluidic chip design enables cost-effective three-dimensional spatiotemporal transcriptomics with a wide field of view. Nat Genet. 2024 Oct;56(10):2259-2270), and DBIT-seq (Liu Y, Yang M, et al. High-Spatial-Resolution Multi-Omics Sequencing via Deterministic Barcoding in Tissue. Cell. 2020 Dec 10;183(6):1665-1681.el8).

The methods described herein have several significant improvements over the above-mentioned platforms, including: i) a way to evenly and reproducibly anchor DNA oligos onto a surface such as a glass slide at high density, and ii) barcoded oligonucleotides that are fully compatible with long-read sequencing (a feature no other spatial or single-cell technology has).

Unless defined otherwise in this specification, technical, and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.

As used throughout this specification and the claims, the terms “comprising”, “containing”, “including”, and its variants are inclusive of other components, elements, integers, steps and the like. Conversely, the term “consisting” and its variants are exclusive of other components, elements, integers, steps and the like.

It is to be noted that the term “a” or “an”, refers to one or more, for example, “an oligonucleotide”, is understood to represent one or more oligonucleotides. As such, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.

As used herein, the term “about” means a variability of plus or minus 10% from the reference given, unless otherwise specified.

As used herein, the phrase “consisting essentially of’ limits the scope of a described composition or method to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the described or claimed method or composition.

Wherever in this specification, a method or composition is described as “comprising” certain steps or features, it is also meant to encompass the same method or composition consisting essentially of those steps or features and consisting of those steps or features.

Compositions

A “nucleic acid”, “nucleic acid sequence”, or “nucleotide sequence” as described herein, can be RNA, DNA, or a modification thereof, and can be single or double stranded. The terms “nucleotide”, “nucleic acid”, “nucleotide residue”, and “nucleic acid residue” are used interchangeably, referring to a nucleotide in a nucleic acid polymer.

Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. As used herein, RNA may be any RNA molecule which may occur in a cell. Thus, it may be mRNA, tRNA, rRNA, viral RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), ribozymal RNA, antisense RNA or non-coding RNA. Preferably, it is mRNA.

As used herein, deoxyribonucleic acid (DNA) is a polymeric molecule formed by deoxyribonucleic acid, including, but not limited to, genomic DNA, double-strand DNA, single-strand DNA, DNA packaged with a histone protein, complementary DNA (cDNA which is reverse-transcribed from an RNA), mitochondrial DNA, and chromosomal DNA.

Nucleic acid sequences described herein can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and/or molecular cloning (e.g. GeneArt, GenScript, Life Technologies, Eurofins).

dNTP stands for deoxyribonucleotide triphosphate. Each dNTP is made up of a phosphate group, a deoxyribose sugar and a nitrogenous base. There are four different dNTPs and can be split into two groups: the purines (including dATP, deoxyadenosine 5'-triphosphate, and dGTP, deoxyguanine 5'-triphosphate) and the pyrimidines (including dTTP, deoxythymidine 5'-triphosphate, and dCTP, deoxy cytidine 5'-triphosphate). As used herein, dNTP Mix (also referred to as dNTPs herein) is a mixture (normally in a solution containing sodium salts) of dATP, dCTP, dGTP and dTTP, suitable for use in polymerase chain reaction (PCR), sequencing, fill-in reactions, nick translation, cDNA synthesis, and TdT-tailing reactions.

As used herein, “complementary DNA” or “cDNA” can refer to a synthetic DNA reverse transcribed from RNA through the action of a reverse transcriptase. The cDNA may be single-stranded or double-stranded and can include strands that have either or both of a sequence that is substantially identical to a part of the RNA sequence or a complement to a part of the RNA sequence.

As used herein, the term “oligonucleotide” or “oligo” refers to short DNA or RNA molecules. In one embodiment, an oligo can be at least about 1 to 500 monomeric components, e.g., nucleotides, in length. In a further embodiment, an oligo can be about 20 to about 80 nucleotides in length. Thus, in various embodiments, an oligo is formed of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 nucleotides. However, in some instances, where the oligo is comprised of several smaller oligos, the resultant oligo may be longer than 100 nucleotides.

Some embodiments include the use of primers. As used herein, a “primer” can refer to a short polynucleotide, generally with a free 3 '-OH group, that binds to a target or template polynucleotide present in a sample by hybridizing with the target or template, and thereafter promoting extension of the primer to form a polynucleotide complementary to the target or template. Primers can include polynucleotides ranging from 5 to 1000 or more nucleotides. In some embodiments, the primer has a length of at least 4 nucleotides, 5 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, or a length within a range of any two of the foregoing lengths.

As used herein, a “barcode” describes a defined polymer, e.g., a polynucleotide, which when it is a functional element of the polymer construct, is specific for a compartment, a single cell, or cell nucleus or cellular components (for example, DNA, RNA and/or mitochondria and ribosomes) thereof. In one embodiment, the barcode is about 2 to 4 monomeric components, e.g., nucleotide bases, in length. In other embodiments, the barcode is at least about 1 to 100 monomeric components, e.g., nucleotides, in length. Thus, in various embodiments, the barcode is formed of a sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99, or up to 100 monomeric components, e.g., nucleotides. In one embodiment, the barcode is lOnt long. A barcode can be an artificial sequence or a naturally occurring sequence. In certain embodiments, each barcode within a population of barcodes is different. In other embodiments, a portion of barcodes in a population of barcodes is different, e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the barcodes in a population of barcodes is different. A population of barcodes may be randomly generated or non-randomly generated. In certain embodiments, a population of barcodes are error correcting barcodes. Barcodes can be used to computationally deconvolute the multiplexed sequencing data and identify sequence reads derived from an individual cell, compartment, etc. A barcode can also be used for deconvolution of a collection of cells or cell nuclei or cellular components thereof that have been distributed into small compartments for enhanced mapping.

In certain embodiments, the term “barcode” and “barcoded” also refers to a process of introducing a barcode to a DNA or RNA. An example of introducing a barcode to an RNA is illustrated in the reverse transcriptase step of FIG. 12 wherein the substrate oligonucleotide is a template for extension of the captured RNA.

The terms “another,” “first,” “second,” “third,” “fourth,” “fifth,” and “sixth,” are used throughout this specification as reference terms to distinguish between various forms and components of the compositions and methods, for example, barcodes.

As used herein, the term “array” or “capture array” refers to a population of oligonucleotides or sites on a solid substrate that can be differentiated from each other according to relative location. Different oligonucleotides that are at different sites or features of an array can be differentiated from each other according to the locations of the sites or features in the array. An individual site or feature of an array can include one or more molecules of a particular type (e.g. species of capture probe). For example, a site or feature can include a single nucleic acid molecule having a particular sequence or a site can include several nucleic acid molecules having the same sequence.

Oligonucleotides may be attached to the solid substrate, e.g. array, of the invention by any suitable means. As used herein, the terms “attached” or “bound” refers to the state of two things being joined, fastened, adhered, connected or bound to each other. For example, a nucleic acid can be attached to a material, such as a gel or solid support, by a covalent or non-covalent bond. A covalent bond is characterized by the sharing of pairs of electrons between atoms. A non-covalent bond is a chemical bond that does not involve the sharing of pairs of electrons and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions and hydrophobic interactions.

As used herein, the term “linker” when referring to the sequences within the oligonucleotides, refers to a short polynucleotide sequence of 2 to 100 nt that is used as a connector between the various components of the substrate oligonucleotides, e.g., the positional barcodes. The terms “solid substrate,” “solid surface” and other grammatical equivalents herein refer to any material that is appropriate for or can be modified to be appropriate for the attachment of materials for the processing of nucleic acids, including, for example, materials for nucleic acid library preparation. As will be appreciated by those in the art, the number of possible solid substrate materials is very large. Possible materials include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, etc.), polysaccharides, nylon or nitrocellulose, ceramics, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, optical fiber bundles, and a variety of other polymers.

In certain embodiments, a solid substrate includes silica-based substrates, such as glass, fused silica, or other silica-containing materials. In certain embodiments, silica-based substrates can also be silicon, silicon dioxide, silicon nitride, or silicone hydrides. In some examples, a solid substrate includes plastic materials such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylons, polyesters, polycarbonates, cyclic olefin polymers, or poly(methyl methacrylate). In certain embodiments, the solid substrate is a silica-based material or plastic material. In certain embodiments, the solid substrate has at least one surface comprising glass.

In certain embodiments, the solid substrate comprises a patterned surface. A “patterned surface” refers to an arrangement of different capture regions in or on an exposed layer of a solid support. In certain embodiments, the pattern can be an x-y format of features that are in rows and columns. In certain embodiments, the pattern can be a repeating arrangement of features and/or interstitial regions. In some examples, the pattern can be a random arrangement of features and/or interstitial regions.

In certain embodiments, a solid substrate is “functionalized”, i.e., coated with a surface polymer comprising functional groups capable of forming covalent bonds with oligonucleotides or modified oligonucleotides, such as those described in PCT Publ. Nos. WO 2013/184796 or WO2016/066586.

In certain embodiments, the solid substrate is a glass slide, functionalized with a surface polymer comprising amino groups, e.g., capable of forming bonds with oligonucleotides that have been functionalized, e.g., on the 5’ end. The solid substrate can be of any size. In certain embodiments, the substrate is a glass slide having standard dimensions of approximately 75mm x 25mm x 1 mm. In certain embodiments, the solid substrate is a slide with a coating to optimize binding of amine-modified oligos, DNA, and/or RNA. In certain embodiments, the slide is coated with a surface polymer that includes an amine-reactive functional group. In certain embodiments, the amine-reactive functional group is N-oxysuccinimide. Examples of coated surfaces include Surmodics TRIDIA™ surface coatings, including TRIDIA™ NHS and TRIDIA™ HD surfaces (available from Surmodics®; Eden Prairie, Minnesota). FIG. 16 provides a fluorescent image of a mi ST array produced using a using a TRIDIA™ coated slide, capture oligos (poly(d)T) were detected probe using a labeled probe.

The term “biological specimen” is intended to mean one or more cell, tissue, organism or portion thereof. It will be evident that a biological specimen from any organism may be used, e.g. plant, animal or fungal. The compositions and methods described herein allow the capture of any nucleic acid, e.g. mRNA molecules present in cells or tissues. The compositions and methods are particularly suitable for isolating and analyzing the transcriptome or genome of cells within a biological specimen, e.g. a tissue sample, wherein spatial resolution of the transcriptomes is desirable, e.g. where the cells are interconnected or in contact directly with adjacent cells. However, it will be apparent to a person of skill in the art that the compositions and methods may also be useful for the analysis of the transcriptome of different cells or cell types within a sample even if said cells do not interact directly, e.g. a blood sample. In other words, the cells do not need to present in the context of a tissue and can be applied to the array as single cells (e.g. cells isolated from a non-fixed tissue, e.g. a blood sample). Such single cells, while not necessarily fixed to a certain position in a tissue, are nonetheless applied to a certain position on the array and can be individually identified. Thus, in the context of analyzing cells that do not interact directly, or are not present in a tissue context, the spatial properties of the described methods may be applied to obtaining or retrieving unique or independent transcriptome information from individual cells.

The biological specimen may be a harvested or biopsied tissue sample, or a cultured sample. Representative samples include clinical samples e.g. whole blood or blood-derived products, blood cells, tissues, biopsies, or cultured tissues or cells etc. including cell suspensions. Artificial tissues may for example be prepared from cell suspension (including for example blood cells). Cells may be captured in a matrix (for example a gel matrix e.g. agar, agarose, etc) and may then be sectioned in a conventional way. Such procedures are known in the art in the context of immunohistochemistry (see, e.g., Andersson, et al 2006, J. Histochem. Cytochem. 54(12): 1413-23. Epub 2006 Sep 6). As described herein, micro isoform spatial transcriptomics (miST) utilizes reverse transcription (RT) primers comprised within substrate oligonucleotides that further comprise unique positional tags (referred to as barcode sequences), which are immobilized on a solid substrate, e.g. a glass slide, to generate an “array”. The unique positional tags correspond to the specific capture region, i.e., location of the substrate oligonucleotide on the array.

Biological specimens, such as tissue sections, are placed onto the array and a reverse transcription reaction is performed with the tissue section on the array. The substrate oligonucleotides, to which the RNA in the tissue sample binds (or hybridizes), are extended using the bound RNA as a template to obtain cDNA. Further, the information from the substrate oligonucleotides is added to the end of the RNA strand. As consequence of the unique positional tags in the substrate oligonucleotides, each cDNA strand and RNA strand carry information about the position of the template RNA in the tissue section. The cDNA-RNA hybrid is then harvested and the RNA is analyzed, e.g. sequenced, which results in a transcriptome with positional information. The sequence data can then be matched to a position in the tissue sample. For instance, the tissue section may be visualized or imaged, e.g. stained and photographed, before or to facilitate the correlation of the positional barcode tags in the RNA molecule with a position within the tissue sample and the sequence data may be overlaid on an image of the tissue specimen, e.g. using computer software, to display the expression pattern of any gene of interest across the tissue. In certain embodiments, a set of fiducial pixels are utilized in this process. However, the visualization step is not essential as the tagged RNA molecule may be correlated with a position in the tissue sample using other means, e.g. the unique profile of the molecules captured with the same positional tag, i.e. at the same location (feature) on the array, may enable the molecule to be correlated to a position within the tissue sample based on the known expression characteristics of cells or areas of cells within the tissue sample.

As used herein, the term “substrate oligonucleotides” refers to the polynucleotides that are affixed to the solid substrate in a specific pattern (array) that provide, inter alia, positional information about the specific capture region to which they are affixed. The substrate oligonucleotides include multiple positional barcodes (e.g., two or more of a first sector barcode, a second sector barcode, a first pixel barcode, and a second pixel barcode), one or more linkers, a cleavable motif, a UMI, and/or a capture sequence. The substrate oligonucleotides have one or multiple regions that are common to all substrate oligonucleotides, for example, a cleavable motif, primer handle and/or capture sequence, and one or more regions that vary depending on the position of the substrate oligo on the substrate, for example, the barcode sequences.

The substrate oligonucleotides are comprised of multiple smaller oligos, that are combinatorially assembled onto the solid substrate surface. Once an oligo is bound to the substrate, it is then referred to as the substrate oligo (or part of the substrate oligo).

The substrate oligos are bound/affixed to the solid substrate via covalent binding with a functional molecule on the solid substrate, such as a primary amine group. The first positional oligonucleotide, which is referred to as the “first sector oligonucleotide” is attached to the substrate using a crosslinker. In certain embodiments, a water-soluble crosslinker is used. In one embodiment, a sulfo-NHS ester is used. In one embodiment, the crosslinker is bis(sulfosuccinimidyl)suberate (BS3), an amine-to-amine crosslinker that is homobifunctional, water-soluble, non-cleavable and membrane impermeable. In certain embodiments, an alternative cross-linking reaction is employed. In certain embodiments, the solid substrate includes an In certain embodiments, a N-oxysuccinimide (NHS ester) is the reactive group (e.g., a TRIDIA™ NHS and TRIDIA™ HD surface). While the first sector oligo is exemplified as the first positional oligo, the order of the positional oligos in the substrate oligonucleotide is not critical. Thus, it is intended that in other embodiments, a different positional oligonucleotide, such as the first pixel oligonucleotide, can be used as the first positional oligonucleotide.

The substrate oligos include one or more of the following smaller positional oligos: a first sector barcode oligo, a second sector barcode oligo, a first pixel barcode oligo, and a second pixel barcode oligo. The substrate oligo also includes a capture sequence, which may be added combinatorially, or may be included at the 3’ end of the last positional oligo. The first sector barcode oligo includes a first sector barcode and a first linker. The first positional oligonucleotide is functionalized using an amino modification at the 5’ end, e.g., amino linker C6 can be used to incorporate an active primary amino group onto the 5'-end of an oligonucleotide. The first positional oligonucleotide further comprises a cleavable motif, optionally a PCR primer handle, and a UMI.

The second sector barcode oligo includes a second sector barcode and a second linker. The first pixel barcode oligo includes a first pixel barcode and a third linker. The second pixel barcode oligo includes a second pixel barcode and a fourth linker. A capture oligo includes a capture sequence, such as a poly(d)T. In another embodiment, the second pixel barcode oligo includes a second pixel barcode and a capture sequence. In another embodiment, the second sector barcode oligo includes a second sector barcode and a capture sequence.

Non-limiting examples of the organization of the components of the substrate oligonucleotides, while allowing for additional intervening elements, include the following: i) a sequence comprising a 5’ amine modification : cleavable motif : UMI : offset UMI-anchor : first sector barcode : linker : second sector barcode : linker : first pixel barcode : linker : second pixel barcode : linker : capture sequence

ii) a sequence comprising a 5’ amine modification : cleavable motif : UMI : offset UMI-anchor : first pixel barcode : linker : second pixel barcode : linker : first sector barcode : linker : second sector barcode : linker : capture sequence

iii) a sequence comprising a 5’ amine modification : cleavable motif : UMI : offset UMI-anchor : first sector barcode : linker : second sector barcode : linker : first pixel barcode : linker : second pixel barcode : capture sequence

iv) a sequence comprising a 5’ amine modification : cleavable motif : UMI : offset UMI-anchor : first pixel barcode : linker : second pixel barcode : linker : first sector barcode : linker : second sector barcode : capture sequence

The other positional barcode oligos are combinatorially added to the first positional barcode oligo in sequence, by using splint oligonucleotides. “Splint oligonucleotide”, as used herein, refers to an oligonucleotide having a sequence that is complementary to a portion of two subsequent sections of the substrate oligonucleotide. For example, referring to FIG. 1, the first splint oligonucleotide contains a sequence complementary to Linker A (the first linker) and barcode SH (second sector barcode). The splint oligonucleotides may be part of a duplex with a barcode oligonucleotide, and have a 3’ overhang that is complementary to the next barcode oligonucleotide. In other embodiments the splint oligonucleotides are added separately from the barcode oligonucleotides.

In certain embodiments, a set of first splint oligonucleotides is provided, wherein each first splint oligonucleotide in the set has a sequence complementary to the first linker and one of the second sector barcodes, whereby the first splint oligonucleotide is bound to the first sector oligonucleotide. In one embodiment, the first sector barcode oligonucleotides comprise a duplex region that comprises a splint oligo, the duplex region having an overhang having a sequence complementary to one of the second sector barcodes. After addition of the second sector barcode oligonucleotide, it is bound to the first splint oligonucleotide, and thus, the substrate. The solid substrate is then contacted with a set of second splint oligonucleotides, each second splint oligonucleotide in the set comprising a sequence complementary to the second linker and one of the first pixel barcodes whereby the second splint oligonucleotide is bound to the second sector oligonucleotide. In one embodiment, the second sector barcode oligonucleotides comprise a duplex region, the duplex region having an overhang having a sequence complementary to one of the first pixel barcodes. After addition of the first pixel barcode oligonucleotide, it is bound to the second splint oligonucleotide, and thus, the substrate. The solid substrate is then contacted with a set of third splint oligonucleotides, each third splint oligonucleotide in the set comprising a sequence complementary to the third linker and one of the second pixel barcodes whereby the third splint oligonucleotide is bound to the first pixel barcode oligonucleotide. In one embodiment, the third sector barcode oligonucleotides comprise a duplex region, the duplex region having an overhang having a sequence complementary to one of the second pixel barcodes. After addition of the second pixel barcode oligonucleotide, it is bound to the third splint oligonucleotide and thus, the substrate. The solid substrate is then contacted with a fourth splint oligonucleotide comprising a sequence complementary to the fourth linker and the capture oligonucleotide whereby the fourth splint oligonucleotide is bound to the second pixel barcode oligonucleotide. In one embodiment, the fourth sector barcode oligonucleotides comprise a duplex region, the duplex region having an overhang having a sequence complementary to the capture sequence. After addition of the capture oligonucleotide, it is bound to the fourth splint oligonucleotide, and thus, the substrate.

The individual barcode oligonucleotides must be joined together. This can be accomplished using a DNA ligase. In certain embodiments, after addition of the final oligonucleotide (e.g., the capture oligonucleotide), a ligation reaction is performed using a DNA ligase. In certain embodiments, a ligation reaction is performed after each addition of subsequent oligonucleotide. In certain embodiments, a T4 ligase is used. Other useful ligases include T3 ligase, T7 ligase, PBCV-1 DNA ligase, or E. coli ligase.

The amount of ligase used can vary. For example, in certain embodiments, 10,000-50,000 units of T4 ligase are utilized. 0.01 Weiss unit of T4 DNA Ligase is the amount of enzyme required to catalyze the ligation of greater than 95% of Ipg of k/Hindlll fragments at 16°C in 20 minutes. In certain embodiments, about 22,500 units of T4 ligase are used.

In alternative embodiments, a hybridization-and-extension approach is used to assemble the substrate oligos. This may be accomplished using a Klenow reaction, which has been described previously. See, e.g., Lbtstedt, B., Strazar, M., Xavier, R. et al. Spatial host- microbiome sequencing reveals niches in the mouse gut. Nat Biotechnol 42, 1394-1403 (2024).. In these embodiments, the splint oligos act as the DNA template for the KI enow reaction. In certain embodiments, a first splint oligonucleotide is provided, wherein the splint oligonucleotide has a sequence complementary to the first linker, whereby the first splint oligonucleotide is bound to the substrate oligonucleotide. A Klenow hybridization-and-extension reaction is then performed. The solid substrate is then contacted with a second splint oligonucleotide, the second splint oligonucleotide comprising a sequence complementary to the second linker, whereby the second splint oligonucleotide is bound to the substrate oligonucleotide. A Klenow hybridization-and-extension reaction is then performed. The solid substrate is then contacted with a third splint oligonucleotide, the third splint oligonucleotide comprising a sequence complementary to the third linker, whereby the third splint oligonucleotide is bound to the substrate oligonucleotide. A Klenow hybridization-and-extension reaction is then performed. The solid substrate is then contacted with a fourth splint oligonucleotide comprising a sequence complementary to the fourth linker, whereby the fourth splint oligonucleotide is bound to the substrate oligonucleotide. A Klenow hybridization-and-extension reaction is then performed. In certain embodiments, the fourth sector barcode oligonucleotides comprise a duplex region, the duplex region having an overhang having a sequence complementary to the capture sequence. After addition of the capture oligonucleotide, it is bound to the substrate oligonucleotide.

The splint oligos are removed using any suitable method, such as incubation with formamide.

The arrays utilized herein are divided into two or more “sectors”, each of which includes a particular section or region of the total array. For example, by using a combination of four first sector barcodes, and four second sector barcodes, a total of 16 sectors are generated and identifiable during downstream sequencing and analysis. For clarity, in this example, the area of the total array (or a portion thereof) is divided into sectors horizontally (along x-axis). Each horizontal section receives the same “first sector” barcode, which is different from the barcodes of the other three sections. The presence of an additional, second barcode in an orthogonal manner divides the area into four sectors vertically (along y-axis). Each vertical section receives the same “second sector” barcode, which is different from the barcodes of the other three sections. The use of the terms “first” and “second” are interchangeable, in that either the “first sector” barcode or “second sector” barcode can be applied first. Thus, in this example, each capture region (sector) will contain one of four unique “first sector” barcodes and one of four unique “second sector” barcodes, totaling 16 unique combinations of first and second sector barcodes (see, e.g., FIG. 1 A - FIG. 1C). While the arrays exemplified herein utilize 16 sectors, greater or fewer sectors can be generated by varying the number of unique first sector and second sector barcodes. In certain embodiments, from about 2 to about 16 first sector barcodes are included in the set. In certain embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 first sector barcodes are included in the set. In certain embodiments, from about 2 to about 16 second sector barcodes are included in the set. In certain embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 second sector barcodes are included in the set. In other embodiments, only a single sector is used, i.e., no sector barcodes are included.

A set of x-axis barcodes (first pixel barcodes) is deposited in rows of capture regions over the full area of the array, such that each of the sectors referred to above receives the same set of first pixel barcodes. A set of y-axis barcodes (second pixel barcode) is deposited in columns of capture regions over the full area of the array, such that each of the sectors referred to above receives the same set of second pixel barcodes. The result is a capture array wherein each sector contains capture regions that include the same first pixel barcodes and second pixel barcodes, but different sector barcodes (see, e.g., FIG. 1 A - FIG. 1C).

It is to be understood that terms such as “vertical” and “horizontal” to describe the organization of sectors, for example, can be interchangeable based on relative orientation. Similarly, the terms “x-axis” and “y-axis” with respect to organization of capture regions, for example, can be interchangeable to arrive at alternate embodiments.

In certain embodiments, from about 2 to about 200 first pixel barcodes are included in the capture array. In certain embodiments, from about 50 to about 100 first pixel barcodes are included in the capture array. In certain embodiments, from about 75 to about 96 first pixel barcodes are included in the capture array. In certain embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 first pixel barcodes are included in the array. The number of first pixel barcodes is constrained by the minimum size of the channel (about 3 pm) and the total size of the array.

In certain embodiments, from about 2 to about 200 second pixel barcodes are included in the capture array. In certain embodiments, from about 50 to about 100 second pixel barcodes are included in the capture array. In certain embodiments, from about 75 to about 96 second pixel barcodes are included in the capture array. In certain embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 second pixel barcodes are included in the array. The number of second pixel barcodes is constrained by the minimum size of the channel (about 3 pm) and the total size of the array.

In certain embodiments, from about 2 to about 20 first sector barcodes are included in the capture array. In certain embodiments, from about 2 to about 10 first sector barcodes are included in the capture array. In certain embodiments, 4 first sector barcodes are included in the array. In certain embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 first sector barcodes are included in the array.

In certain embodiments, from about 2 to about 20 second sector barcodes are included in the capture array. In certain embodiments, from about 2 to about 10 second sector barcodes are included in the capture array. In certain embodiments, 4 second sector barcodes are included in the array. In certain embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second sector barcodes are included in the array.

In certain embodiments, a capture array contains at least 5000, 10000, 20000, 40000, 50000, 75000, 100000, 150000, 200000, 300000, 400000, 500000, 750000, 800000, 1000000, 1200000, 1500000, 1750000, or 18000000 distinct capture regions. The relative size of capture regions and/or average distance between capture regions may be decreased to allow greater numbers of capture regions to be accommodated within the same or a similar area. Furthermore, the number of sectors can be increased to allow for greater numbers of capture regions within the same or a similar area. In certain embodiments, the arrays provided herein can accommodate up to about 9,200 unique capture regions in an area of about 9.2 mm², for example in a 96x96 channel device (100pm), with a single sector. In certain embodiments, the arrays provided herein can accommodate up to about 1,800,000 unique capture regions in an area of about 8.0 mm², for example in a 96x96 channel device (3 pm), having 14x14 sectors.

The substrate oligo includes multiple barcode sequences that, together, provide spatial location information for the capture region and the RNA molecule attached thereto. Each barcode can be designed to any length available using synthesis technology. For example, in one embodiment, each barcode is between 5 nt to 100 nt in length. In another embodiment, the barcode sequences are between 10 nt to 20 nt in length. In one embodiment, the barcode is 10 nt in length. In another embodiment, the barcode is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nt in length.

The design of barcode sequences can be optimized to improve downstream sequencing. In certain embodiments, the sector and pixel barcode sequences are designed in a combinatorial, error-robust manner. For example, the barcode sequences can be designed to lack DNA base repeats and/or homopolymeric regions. In certain embodiments, the barcode sequences of an array all have about 50% GC content. In certain embodiments, the barcode sequences are designed to have bases at their 5’ and 3’ ends that are different from adjacent bases of intervening linker sequences (i.e., degenerate bases to remove possibility of base repeats in downstream sequencing). In certain embodiments the barcodes have one or more of the following characteristics: lOnt in length, have 50% GC content, and contain no homopolymer repeats. In addition, in certain embodiments, such as for the barcodes exemplified herein, the shared k-mer length has been minimized and the pairwise distance (tested for Levenshtein and Hamming) has been maximized (see FIG. 9).

The table below provides an exemplary set of sector and pixel barcodes for generating substrate oligonucleotides for a 4x4 array having 16 sectors. SEQ SEQ

SEQ ID SEQ ID ID sector_l ID sector_2 bc_x bc_y

NO: NO:

NO: NO:

AGCACGAT AGCATA

1 5 9 ACACACGTGA 105 CACGTCATCA CT GCGT ATCACGAC CAGTGT

2 6 10 ACACTAGAGC 106 CACTCGAGTA AC CACA GCGATATC TCAGAC

3 7 11 ACAGTCACGA 107 CAGAGCAGTA GT AGTG GTATGCGA TGCGTG

4 8 12 ACATAGCACG 108 CAGCTACGTA CT TGTA

13 ACATCAGTCG 109 CATACTGCAC 14 ACGACAGTAC 110 CATCAGCATG 15 ACGAGATAGC 111 CATCGTCTAC 16 ACGAGTACAC 112 CATCTGACGA 17 ACGATCGACA 113 CATGATGAGC 18 ACGATGTCAG 114 CGACACGATA 19 ACGCATATGC 115 CGACGTACTA 20 ACGCTGATCA 116 CGACTATACG 21 ACGTCGTACA 117 CGATCGATCA 22 ACTACTGCTG 118 CGTCAGTAGA 23 ACTCTCACTC 119 CGTGTGCATA 24 ACTGCGATGA 120 CTAGCAGAGA 25 AGACAGAGAC 121 CTCAGTACTG 26 AGACTAGCTG 122 CTCAGTGTCA 27 AGAGCACATC 123 CTCATACGCA 28 AGATGCGTAC 124 CTCGACAGTA 29 AGCAGATGTG 125 CTCGATGTAC 30 AGCATAGTGC 126 CTCGTGATAG 31 AGCTCATGAC 127 CTCTACGATC 32 AGCTGTATGC 128 CTCTCTGCTA 33 AGTAGCAGTC 129 CTCTGATCGA 34 AGTCAGACTG 130 CTGACTAGCA 35 AGTCGTGCTA 131 CTGATGCAGA 36 AGTGCTCACA 132 GACACTAGAC 37 AGTGTGTGAG 133 GACGCATGTA 38 ATAGCGCTCA 134 GACTCGATAG 39 ATATCGAGCG 135 GACTGTACGA 40 ATCGTCTCTC 136 GAGACACAGA 41 ATCTGTCGCA 137 GAGCTATCGA 42 ATGCAGTGTC 138 GAGTCATGAC 43 ATGTACGCAG 139 GCACATACGA 44 ATGTCGACGA 140 GCACATCTAC 45 CACACTCGTA 141 GCAGAGTATC 46 CACAGATCTC 142 GCAGATGACA 47 CACGTCAGTA 143 GCATAGTGCA 48 CAGCTATGTC 144 GCATCTATGC 49 CAGTCTGTAC 145 GCATGCACTA 50 CAGTGCACTA 146 GCGATATACG

51 CATACGTGCA 147 GCTATGCACA CATAGACGAC 148 GCTCATGATC CATGTGCATG 149 GCTGATATCG CGACATCTCA 150 GTACGTCACA CGAGACATAC 151 GTAGTCATCG CGATACTCGA 152 GTCAGCTCTA CGATCATGCA 153 GTGACATGTG CGCAGTATAG 154 GTGTGATCTC CGCGATATGA 155 TACACGACTC CGCTAGTGTA 156 TACGACTGTC CGTACTCATC 157 TACGTGAGAC CGTAGCTAGA 158 TACTCACGAG CGTATGATGC 159 TACTGTCGCA CGTCTACGTA 160 TACTGTGCAC CTACAGTGAG 161 TAGAGTGAGC CTACGCTATC 162 TAGCACTGCA CTAGATCACG 163 TAGCAGCAGA CTAGTACGTC 164 TAGCGATCAC CTAGTCGCTA 165 TAGCTGACTG CTATCGCATC 166 TAGTAGCGTG CTATGAGCTC 167 TAGTGCGTAG CTCTATCAGC 168 TATCGCGATG CTCTGAGTGA 169 TATCTAGCGC CTGACATGAG 170 TATGCGATCG CTGAGACGTA 171 TATGCGTCAC CTGCTCTACA 172 TCACGCATAC CTGTCTAGCA 173 TCAGACAGTG GACTACACTG 174 TCAGATCAGC GACTCTAGAC 175 TCAGCGTCTA GACTGTCATC 176 TCAGCTGATG GAGATCGTAG 177 TCATACTCGC GAGTAGTCTC 178 TCGACATCTC GATCAGAGTC 179 TCGACTAGAG GATGATCTGC 180 TCGCTAGTAC GCACTGATAC 181 TCGCTCTCTA GCATACTGAG 182 TCGTCGATAC GCATCACATG 183 TCGTCGTACA GCATCTACTC 184 TCGTGAGCTA GCGCATGATA 185 TCTAGTCGAC GCGTGATGTA 186 TCTGAGCTCA GCTATACAGC 187 TCTGTCGACA GCTGACTATC 188 TCTGTGACTC GTACATGCGA 189 TGACTCTAGC GTACGACTGA 190 TGACTGCTCA GTACTGTAGC 191 TGATCAGACG GTAGTGATCG 192 TGCATAGCTC GTATCTCGAG 193 TGCATCTGTG GTCATACACG 194 TGCGCATATC GTCGATAGAG 195 TGCTCTACAG GTCGCGTATA 196 TGCTGATCTG

GTCTGATGAG 197 TGTAGCTCTC 102 GTCTGCTACA 198 TGTCAGAGTC 103 GTGACTCAGA 199 TGTCGATGCA

104 GTGTAGAGTG 200 TGTGACGATC

An oligonucleotide may be cleavable, for example, to release is from a solid substrate. In certain embodiments, the oligonucleotides provided herein comprise one or more cleavable motifs. In certain embodiments, the cleavage motif is an enzymatic cleavage site, such an endonuclease or restriction recognition site present in a specific nucleic acid sequence. In still other embodiments, a cleavage site is a site of chemical cleavage. In a particular embodiment, the cleavable motif comprises a d(U) cleavable motif. In certain embodiments, the cleavable motif is a sequence of at least 3, 4, 5, or 6 deoxyuridine (dU) bases.

The substrate oligonucleotides include a “unique molecular identifier” (UMI), which is a random sequence of nucleotide bases. The UMI is sequencing linker or a subtype of nucleic acid barcode that permits identification of amplification duplicates of the polymer construct/construct oligonucleotide sequence with which it is associated. The UMI may also be used to determine the number of transcripts that gave rise to an amplified product. One or more UMI may be included in a construct oligonucleotide sequence. In certain embodiments, the UMI is positioned 5’ of barcode sequence in a nucleotide construct (e.g., as depicted in FIG. 8). In certain embodiments, the UMI is about 8 to about 12 nucleotides in length. In other embodiments, each UMI can be at least about 1 to 100 nucleotides, in length. Thus in various embodiments, the UMI is formed of a random sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 monomeric components, e.g., nucleic acids. In certain embodiments, the UMI is unique to each sector barcode included in a capture array. In certain embodiments, the UMI sequence includes degenerate bases at the 5’ and 3’ ends to determine the start and stop of the UMI sequence.

Further, in certain embodiments, substrate oligonucleotides include an “offset UMI-anchor” to improve the efficiency and quality of downstream sequencing analysis. The offset UMI anchor can be located 3’ of a UMI sequence (e.g., as depicted in FIG. 8). The offset UMI anchor of the substrate oligonucleotides may include a mixture of lengths of offset UMI anchors to facilitate downstream sequencing by preventing sequences from having the same base at a specific position in any given sequencing cycle. By including offset UMI anchors of different lengths, the sequences including different offset UMI anchors are staggered compared to one another by one, two, three, four, five, six, or more base pairs.

The term “PCR handle” refers to a primer binding site cable of being bound by a primer to facilitate PCR amplification. The PCR handle has a length of at least 4 nucleotides, 5 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, or a length within a range of any two of the foregoing lengths. In certain embodiments, the PCR handle is adaptable or specific to one or more sequencing platforms and/or library preparation strategies, including commercially available technologies (e.g., Illumina and 10X Genomics sequencing). In certain embodiments, the PCR handle is absent.

The substrate oligonucleotides include a capture sequence that is capable of hybridizing with RNA present in a biological specimen. The term “capture sequence” refers to a nucleotide that is complementary to and capable of binding a sequence of a target nucleic acid (e.g., an RNA polyA tail). In certain embodiments, the capture sequence includes a poly (d)T sequence. In certain embodiments, the capture sequence is a poly(d)T VN sequence (i.e. a sequence having a string of thymidine residues followed by dV (either dG, dA, or dC) and then by dN (dA, dT, dG, or dC). Still other capture sequences may be employed for capture of specific target sequences or target RNA species. In certain some embodiments, the capture sequence is positioned at the 3’ end of a substrate oligonucleotide. In certain embodiments, the capture sequence has a length of at least 4 nucleotides, 5 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, or a length within a range of any two of the foregoing lengths.

In certain embodiments, the capture array includes a series of regions or spots with one or more bound oligonucleotides termed “fiducial pixels” that aid in accurate assignment of sequences to capture regions and corresponding histological features of the biological specimens. In certain embodiments, the fiducial pixels are patterned in a row, a column, or a grid on the solid substrate. In certain embodiments, the fiducial pixels may be interspersed among the capture regions of bound oligonucleotides, for example throughout one or more sectors of the array. In certain embodiments, the capture array includes fiducial pixels outside of the sector area but within a field of a biological specimen to be imaged. In certain embodiments, the fiducial pixels are oligonucleotides bound to the substrate that can be labeled by contacting them with a complementary oligonucleotide that is capable of hybridizing with a bound oligonucleotide and which is labeled with a fluorophore. In certain embodiments, the fiducial pixels are oligonucleotides bound to the substrate that are directly labeled with a fluorophore.

Methods

The present application provides improved methods for generation of capture arrays as well as improved methods for performing spatial transcriptomics.

As described herein, the use of a water-soluble amine linkers (e.g., bis(sulfosuccinimidyl)suberate (BS3)) to conjugate nucleic acids to a substrate, such as a glass surface, can provide certain advantages, including the ability to generate a capture surface of even density (see, for example FIG. 5). The concentration of oligonucleotide can also determine efficiency and density of binding. An exemplary formulation for binding oligos to a substrate is: 2.5mM BS3, 1-lOpM DNA oligo and lx phosphate buffered saline (PBS).

As used herein, the term “amine-reactive crosslinker” or amine-reactive linker” refers to a chemical reagent that covalently bonds to amine groups, typically found in proteins, peptides, and other biological molecules. These crosslinkers contain functional groups capable of reacting with the nucleophilic amine groups present on lysine residues or the N-termini of proteins and other biomolecules. The core utility of amine-reactive crosslinkers lies in their ability to introduce covalent modifications, facilitating the development of stable conjugates.

As described herein, amine reactive linkers are useful in binding oligonucleotides, particularly oligonucleotides have a 5’ amine, to a solid substrate. In certain embodiments, the amine-reactive linker is an N-hydroxysuccinimide (NHS) ester, a sulfo-NHS ester, an isocyanate, an epoxide, an aldehyde, or carboxyl-containing crosslinker. In certain embodiments, the amine-reactive linker is water soluble. In certain embodiments, the linker is bis(sulfosuccinimidyl)suberate (BS3; CAS Number 82436-77-9).

In certain embodiments, a formulation for binding oligonucleotides to a solid substrate comprises a concentration of an amine-reactive linker that is about 0.1 mM to about 10 mM. In certain embodiments, the concentration of an amine-reactive linker is about 0.1 mM to about 8 mM, about 0.1 mM to about 6 mM, about 0.1 mM to about 4 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, about 2 mM to about 3 mM, or about 2 mM to about 4 mM. In certain embodiments, the concentration of an amine-reactive linker is about 2.5 mM.

In certain embodiments, a formulation for binding oligonucleotides to a solid substrate comprises a concentration of oligonucleotides that is about 0.01 pM to about 50 pM. In certain embodiments, the concentration of oligonucleotides is about 0.01 pM to about 50 pM, about 0.01 pM to about 40 pM, about 0.01 pM to about 30 pM, about 0.01 pM to about 25 pM, about 0.01 pM to about 20 pM, about 0.01 pM to about 15 pM, about 0.01 pM to about 10 pM, about 0.1 pM to about 50 pM, about 0.1 pM to about 40 pM, about 0.1 pM to about 30 pM, about 0.1 pM to about 25pM, about 0.1 pM to about 20 pM, about 0.1 pM to about 15 pM, or about 0.1 pM to about 10 pM.

Without intending limit the scope of the present disclosure, the following provides an exemplary method for binding oligonucleotides to a functionalized glass surface using an amine-reactive linker: 1) Contact a slide surface with a formulation that is 2.5mM BS3, 1-lOpM DNA oligo and lx phosphate buffered saline (PBS), and incubating at 25°C for Ihr. 2) Wash the surface area with 2X Saline-sodium citrate (SSC) twice, followed by a wash with 2X SSC + 0.1% SDS (sodium dodecyl sulfate), and 3x washes with Milli-Q® water. 3) Block slides at room temperature for 40min with 50mL of blocking mix: 8g succinic anhydride, ~49.5ml dimethyl formamide and 500 pL triethylamine. 4) Rinse slides 3x with ~40ml Milli-Q® water and spin dry.

In one aspect, provided herein is a method of spatially resolved transcriptome sequencing of one or more biological specimens. In certain embodiments, the methods comprises steps including one or more of a) providing the solid substrate described herein having oligonucleotides bound to a surface; b) mounting the one or more biological samples to the solid substrate, the biological samples partially or completely overlaying the array of capture regions; c) performing staining or immunofluorescent labeling of the one or more biological samples; d) capturing one or more images of the one or more biological samples; e) permeabilizing the one or more biological specimens thereby permitting RNA present in the one or more biological specimens to bind capture sequences of the substrate oligonucleotides; f) performing reverse-transcription; and/or g) sequencing RNA. In certain embodiments, the method further comprises a step wherein the surface and bound oligonucleotides are contacted with an enzyme that cleaves a motif of the oligonucleotides. In certain embodiments, the method further comprises a step wherein the surface and bound oligonucleotides are contacted with an enzyme that cleaves a motif of the oligonucleotides. In certain embodiments, the method further comprises steps to bind, detect, and/or image fiducial pixels. Including fiducial pixels in the array allows for accurate registration of all produced pixel channels simultaneously or in combination with tissue staining, as well as quality control of the produced spatial array and fast concatenation of barcode oligonucleotides within or outside the sampled tissue area (see, e.g., FIG. 7).

The mode of preparation of the biological specimen, e.g. tissue sample, and how the resulting sample is handled may affect the transcriptomic analysis of the methods herein. Moreover, various biological specimens, e.g. tissue samples, will have different physical characteristics and it is well within the skill of a person in the art to perform the necessary manipulations to yield a biological specimen, e.g. tissue sample, for use herein. However, it is evident from the disclosure herein that any method of sample preparation may be used to obtain a biological specimen, e.g. tissue sample, that is suitable for use herein. For instance, any layer of cells with a thickness of approximately 1 cell or less may be used. In one embodiment, the thickness of the biological specimen, e.g. tissue sample, may be less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1 of the cross-section of a cell. In certain embodiments, the methods allow for single cell resolution and the biological specimen, e.g. a tissue sample having a thickness of one cell diameter or less. In certain embodiments, thicker biological specimens, e.g. tissue samples, are used. For example, cryostat sections may be used, which may be e.g. 10-20 pm thick.

The thickness of the biological specimen, e.g. tissue sample section, for use herein may be dependent on the method used to prepare the sample and the physical characteristics of the tissue. Thus, any suitable section thickness may be used. In certain embodiments, the thickness of the tissue sample section will be at least 0.1 pm, further preferably at least 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10pm. In other embodiments the thickness of the tissue sample section is at least 10, 12, 13, 14, 15, 20, 30, 40 or 50pm. However, the thickness is not critical and these are representative values only. Thicker samples may be used if desired or convenient e.g. 70 or 100 pm or more. Typically, the thickness of the tissue sample section is between 1-100 pm, 1-50 pm, 1-30 pm, 1-25 pm, 1-20 pm, 1-15 pm, 1-10 pm, 2-8pm, 3-7pm or 4-6pm, but as mentioned above thicker samples may be used.

The biological specimen, e.g. tissue sample, may be prepared in any convenient or desired way. Fresh, frozen, fixed or unfixed tissues may be used. Any desired convenient procedure may be used for fixing or embedding the biological specimen, e.g. tissue sample, as described and known in the art. Thus, any known fixatives or embedding materials may be used.

In certain embodiments, a tissue is prepared by deep freezing at a temperature suitable to maintain or preserve the integrity (i.e. the physical characteristics) of the tissue structure, e.g. less than -20°C and preferably less than -25, -30, -40, -50, -60, -70 or -80 °C. The frozen tissue sample may be sectioned, i.e. thinly sliced, onto the solid substrate, e.g. array surface by any suitable means. For example, a tissue sample may be prepared using a chilled microtome, a cryostat, set at a temperature suitable to maintain both the structural integrity of the tissue sample and the chemical properties of the nucleic acids in the sample, e.g. to less than -15°C and preferably less than -20 or -25°C. Thus, the sample should be treated so as to minimize the degeneration or degradation of the nucleic acid, e.g. RNA, in the tissue. Such conditions are well-established in the art and the extent of any degradation may be monitored through nucleic acid extraction, e.g. total RNA extraction, and subsequent quality analysis at various stages of the preparation of the tissue sample.

In certain embodiments, a tissue is prepared using standard methods of formalinfixation and paraffm-embedding (FFPE), which are well-established in the art. Following fixation of a tissue sample and embedding in a paraffin or resin block, the tissue sample may be sectioned, i.e. thinly sliced, onto the solid substrate, e.g. array. As noted above, other fixatives and/or embedding materials can be used.

In certain embodiments, the biological specimen, e.g. tissue sample section, will need to be treated to remove the embedding material e.g. to deparaffinize, i.e. to remove the paraffin or resin, from the sample prior to carrying out the methods. This may be achieved by any suitable method and the removal of paraffin or resin or other material from tissue samples is well established in the art, e.g. by incubating the sample (on the surface of the solid substrate, e.g. array) in an appropriate solvent e.g. xylene, e.g. twice for 10 minutes, followed by an ethanol rinse, e.g. 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2 minutes.

In certain embodiments, the nucleic acid molecules of the specimen (particularly RNA molecules) are not modified prior to contact with the solid substrate (e.g. array). For instance, in some preferred embodiments the nucleic acid molecules (e.g. RNA) of the biological specimen are not treated to enhance or facilitate their interaction with the substrate oligonucleotides prior to contact with the solid substrate. In some embodiments, the nucleic acid molecules (e.g., RNA) of the biological specimen are not contacted with or hybridized to nucleic acids (e.g,. aptamers, oligonucleotides or nucleic acid tags) that facilitate the interaction between the nucleic acid molecules of the biological sample and substrate oligonucleotides) prior to contacting the biological specimen with the solid substrate. For instance, in some embodiments, the biological specimen is not contacted with or hybridized to nucleic acids that function as an intermediary between the target nucleic acids of the biological specimen and the substrate oligonucleotides, i.e. that function to indirectly attach or link the target nucleic acid of the biological specimen to the capture probe, prior to contacting the biological specimen with the capture array.

As used herein, the term “hybridize” is used in its broadest sense to mean the formation of a stable nucleic acid duplex. A duplex can be “perfectly matched”, such that the polynucleotide and/or oligonucleotide strands making up the duplex form a double stranded structure with one another such that every nucleotide in each strand undergoes Watson-Crick base pairing with a nucleotide in the other strand. A duplex can comprise at least one mismatch, wherein the term “mismatch” means that a pair of nucleotides in the duplex fail to undergo Watson-Crick bonding. Thus, two sequences need not have perfect homology to be “complementary” under the invention. Usually two sequences are sufficiently complementary when at least about 90% (preferably at least about 95%) of the nucleotides share base pair organization over a defined length of the molecule.

On contact of the biological specimen, e.g. tissue sample section, with the solid substrate, e.g. following removal of the embedding material, e.g. deparaffinization, the nucleic acid, e.g. RNA, molecules in the biological specimen, e.g. tissue sample, will be capable of binding to the immobilized substrate oligonucleotides on the array. In certain embodiments, it may be advantageous to facilitate the hybridization of the nucleic acid, e.g. RNA, molecules to the substrate oligonucleotides. Typically, facilitating the hybridization comprises modifying the conditions under which hybridization occurs. The primary conditions that can be modified are the time and temperature of the incubation of the biological specimen, e.g. tissue sample section, on the array prior to the reverse transcription step, which is described elsewhere herein. In certain embodiments, a cleavage step, as described elsewhere herein, facilitates interactions between substrate oligonucleotides and RNA of the biological sample. In certain embodiments, hybridization occurs at 37-42°C for 30 minutes to 20 hours.

It will be evident that biological specimens, e.g. tissue samples, from different sources may require different treatments to allow the nucleic acids to interact with, i.e. hybridize to, the substrate oligonucleotides. For instance, it may be useful to permeabilize the biological specimen, e.g. tissue sample, to facilitate the interaction between the nucleic acid and the substrate oligonucleotides. If the tissue sample is not permeabilized sufficiently the amount of nucleic acid captured by the substrate oligonucleotides may be too low to enable further analysis. Conversely, if the biological specimen, e.g. tissue sample, is too permeable, the nucleic acid may diffuse away from its origin in the biological specimen, e.g. tissue sample, i.e. the nucleic acid may be captured by the substrate oligonucleotides may not correlate accurately with its original spatial distribution in the biological specimen, e.g. tissue sample. Hence, there must be a balance between permeabilizing the biological specimen, e.g. tissue sample, enough to enable efficient interaction between the nucleic acids and the substrate oligonucleotides while maintaining the spatial resolution of the nucleic acid distribution in the biological specimen, e.g. tissue sample. The methods used to fix the biological specimen, e.g. tissue sample, may also impact on the interaction between the nucleic acid of the biological specimen, e.g. tissue sample, and the substrate oligonucleotides.

Thus, the methods comprise a step of permeabilizing the biological specimen. This step may be performed after the step of contacting the specimen with the solid substrate. Suitable methods and agents for permeabilizing and/or fixing biological specimens, e.g. cells and tissues, are well known in the art and any appropriate method may be selected for use herein. Some proteases are particularly useful in permeabilizing cells in a biological specimen, e.g. pepsin. Particularly useful fixatives include, e.g. methanol.

In order to correlate the sequence analysis or transcriptome information obtained from each capture region of the array with the region (i.e. an area or cell) of the biological specimen, e.g. tissue sample, the biological specimen is oriented in relation to capture regions on the array. In certain embodiments, this step includes visualization of the fiducial pixels. In other words, the tissue sample is placed on the array such that the position of a substance oligonucleotides on the array may be correlated with a position in the biological specimen, e.g. tissue sample. Thus it may be identified where in the biological specimen, e.g. tissue sample, the position of each species of capture probe (or each feature of the array) corresponds. In other words, it may be identified to which location in the biological specimen, e.g. tissue sample, the position of each capture region.

In certain embodiments, the biological specimen, e.g. tissue sample, may be imaged following its contact with the array. In certain embodiments, the biological specimen, e.g. tissue sample, is imaged prior to substrate oligonucleotide cleavage and/or reverse transcription steps. Generally speaking, imaging may take place at any time after contacting the biological specimen, e.g. tissue sample, with the solid substrate, but before any step which degrades or removes the biological specimen, e.g. tissue sample. As noted above, this may depend on the biological specimen, e.g. tissue sample.

As described herein, improvements in the reverse transcription reaction allow for efficient cDNA synthesis. Reverse transcription directly barcodes spatially captured RNA, through DNA-templated extension, wherein substrate oligonucleotides provide a barcode template. These steps are required to enable long read sequencing of RNA without the need for cDNA amplification.

The methods described herein include a reverse transcription step, particularly reverse transcription using an ultra-processive reverse transcriptase. A reverse transcriptase is an enzyme that uses RNA as a template for DNA synthesis. Without wishing to be bound by theory, the ultra-processive reverse transcriptase has helicase activity that allows for processing of longer nucleic acids. Reverse transcriptases useful herein include UltraMarathonRT (uMRT)(RNAConnect), MMLV reverse transcriptase (RT), Maxima H Minus Reverse Transcriptase, SuperScript™ II Reverse Transcriptase (Oscorbin IP, Filipenko ML. M-MuLV reverse transcriptase: Selected properties and improved mutants. Comput Struct Biotechnol J. 2021 Nov 22;19:6315-6327. doi: 10.1016/j.csbj .2021.11.030. PMID: 34900141; PMCID: PMC8640165), Induro® Reverse Transcriptase, or UltraMarathonRT (RNA Connect) (Zhao C, Pyle AM. Crystal structures of a group II intron maturase reveal a missing link in spliceosome evolution. Nat Struct Mol Biol. 2016 Jun;23(6):558-65. doi: 10.1038/nsmb.3224. Epub 2016 May 2. PMID: 27136328; PMCID: PMC4899126).

The following is an overview of exemplary conditions suitable for performing reverse transcription reaction on an array surface - RT reaction is loaded with IX RT buffer, 0.2-0.4 mg BSA, 0.5-1 pmol dNTPs, 5-15 nmol template switch oligo, 0.1X PEG 8000 (50%w/v), 8ul RT Maxima H- enzyme and 4ul RNAOUT (Invitrogen) inhibitor. The reaction volumes are 80 pl. Reaction conditions were run overnight at 42°C for optimal results.

As used herein, “sequencing” generally refers to determining the order of nucleotides (base sequences) in a nucleic acid sample, e.g., DNA or RNA. Many techniques are available, such as Sanger sequencing or High Throughput Sequencing technologies (HTS). Sanger sequencing may involve sequencing via detection through (capillary) electrophoresis, in which up to 384 capillaries may be sequence analyzed in one run. High throughput sequencing involves the parallel sequencing of thousands or millions or more sequences at once. HTS can be defined as Next Generation sequencing (NGS), i.e. techniques based on solid phase pyrosequencing or as Next-Next Generation sequencing based on single nucleotide real time sequencing (SMRT). HTS technologies are available such as offered by Roche, Illumina and Applied Biosystems (Life Technologies). Further high

throughput sequencing technologies are described by and/or available from Helicos, Pacific Biosciences, Complete Genomics, Ion Torrent Systems, Oxford Nanopore Technologies, Nabsys, ZS Genetics, GnuBio. Sequencing platforms such as Nanopore direct RNA sequencing (DRS) allow direct sequencing of full-length native RNA molecules without the need for RT or amplification.

As used herein, “next generation sequencing” refers to high-throughput sequencing methods that allow the sequencing of millions to billions of molecules in parallel. Examples of next generation sequencing methods include sequencing by synthesis, sequencing by ligation, sequencing by hybridization, polony sequencing, ion semiconductor sequencing, nanopore sequencing, and pyrosequencing. By attaching primers to a solid substrate and a complementary sequence to a nucleic acid molecule, a nucleic acid molecule can be hybridized to the solid substrate via the primer and then multiple copies can be generated in a discrete area on the solid substrate by using polymerase to amplify (these groupings are sometimes referred to as polymerase colonies or polonies). Consequently, during the sequencing process, a nucleotide at a particular position can be sequenced multiple times (e.g., hundreds or thousands of times) — this depth of coverage is referred to as

“deep sequencing.” Examples of high throughput nucleic acid sequencing technology include platforms provided by Illumina, BGI, Qiagen, ThermoFisher, and Roche, including formats such as parallel bead arrays, sequencing by synthesis, sequencing by ligation, capillary electrophoresis, electronic microchips, “biochips,” microarrays, parallel microchips, and single-molecule arrays, as reviewed by Service (Science 311:1544-1546, 2006).

In certain embodiments, sequencing comprises detecting and/or distinguishing unmodified and modified nucleobases. For example, Oxford Nanopore direct-RNA sequencing has been shown to be sensitive to RNA modifications (see, e.g., Leger A, et al. Nat Commun. 2021 Dec 10; 12(1):7198. doi: 10.1038/s41467-021-27393-3. PMID:

34893601; PMCID: PMC8664944) Oxford nanopore sequencing systems (e.g., MinlON sequencer) that can directly detect methylation of RNA (for example: an m6A modification) can be used here. Sequencing reactions can be performed in a variety of sample processing units, which may multiple lanes, multiple channels, multiple wells, or other mean of processing multiple sample sets substantially simultaneously. Sample processing unit can also include multiple sample chambers to enable processing of multiple runs simultaneously. Similarly, Ion Torrent sequencing may also be used to directly detect methylation. Thus, in some embodiments, methylation status can be determined during sequencing, e.g., without or independently of a partitioning step or a conversion procedure such as bisulfite treatment.

In certain embodiment, sequencing is performed using nanopore sequencing (e.g. as described in Soni GV and Meller A. Clin Chem 53: 1996-2001, 2007). Nanopore sequencing is a single-molecule sequencing technology whereby a single molecule of RNA is sequenced directly as it passes through a nanopore. The technology allows for characterization and quantification of full-length RNA transcripts, splice variants, and fusions using short to ultralong fragment sequencing A nanopore is a small hole, of the order of 1 nanometer in diameter. Immersion of a nanopore in a conducting fluid and application of a potential (voltage) across it results in a slight electrical current due to conduction of ions through the nanopore. The amount of current which flows is sensitive to the size and shape of the nanopore. As an RNA molecule passes through a nanopore, each nucleotide on the RNA molecule obstructs the nanopore to a different degree, changing the magnitude of the current through the nanopore in different degrees. Thus, this change in the current as the RNA molecule passes through the nanopore represents a reading of the RNA sequence. See, e.g, WO 2016059436 Al, which is incorporated herein by reference.

In certain embodiments, sequencing comprises a step of adapter ligation to nucleic acids to be sequenced. An “adapter” as referred to herein, generally refers to a short nucleic acid molecule (e.g., about 10 to about 100 base pairs in length). An adapter may comprise a short double-stranded DNA molecule. An adapter may be attached, e.g., via polymerization or ligation, to an end of a DNA fragments or amplicons. Adapters may comprise synthetic oligonucleotides, e.g., oligonucleotides that have nucleotide sequences which are at least partially complementary to each other. An adapter may have blunt ends, may have staggered ends (also referred to herein as a 3' or 5' “overhang sequence” or “sticky end”, or a blunt end and a staggered end. Adapters may be attached (e.g., via ligation) to fragments to provide an adapter-ligated fragment; the adapter-ligated fragment may serve as a starting point for subsequent manipulation e.g., for amplification or sequencing. An adapter may be functionalized, e.g., conjugated with a tag, probe, detectable label, affinity capture reagent (e.g., biotin or streptavidin). In certain embodiments, the adapter is an oligonucleotide that is loaded with a motor protein, such as that required for nanopore sequencing.

In certain embodiments, the methods comprise releasing oligonucleotides (including any associated nucleic acids) bound to a substrate. In certain embodiments, the oligonucleotides are released using an enzyme that cleaves a cleavable motif of the oligonucleotides. Those of skill in the art can identify enzymes and the corresponding nucleic acid sequences that are recognized and cleaved. “Cleave” is defined broadly herein to include any means of breaking a covalent bond. In some embodiments, cleavage involves cleavage of a covalent bond in a nucleotide chain (i.e. strand cleavage or strand scission), for example by cleavage of a phosphodiester bond. For example, where the cleavable motif is a series of deoxyuridine (dU) bases, addition of USER enzyme (a mixture of E. coli uracil DNA glycosylase and endonuclease VIII) can be used to release the oligonucleotides. One of skill in the art will recognize release of the oligonucleotides can be achieved using available restriction site targeting enzymes. In certain embodiments, the addition on an enzyme to release the oligonucleotides can be performed prior to or subsequent to RT. In certain embodiments, the enzyme is included in the RT step. In certain embodiments, the cleavage step is performed prior to any RT step to improve RT efficiency.

As described in more detail below, any method of nucleic acid analysis may be used to analyze sequence information obtained according to the disclosed methods. Typically, the sequence analysis information obtained is used to obtain spatial information as to the RNA in the biological specimen, e.g. tissue sample. In other words, the sequence analysis information may provide information as to the location of the RNA in the biological specimen, e.g. tissue sample. This spatial information may be derived from the nature of the sequence analysis information determined, for example it may reveal the presence of a particular RNA which may itself be spatially informative in the context of the biological specimen, e.g. tissue sample, used, and/or the spatial information (e.g. spatial localization) may be derived from the position of the biological specimen, e.g. tissue sample, on the solid substrate, e.g. array, coupled with the sequencing information. Thus, the method may involve simply correlating the sequence analysis information to a position in the biological specimen, e.g. tissue sample, e.g. by virtue of barcoding and its correlation to a position in the biological specimen, e.g. tissue sample. However, in some embodiments, spatial information may conveniently be obtained by correlating the sequence analysis data to an image of the biological specimen, e.g. tissue sample. Accordingly, in a preferred embodiment the method also includes a step of: correlating sequence analysis information with an image of the biological specimen, It will be seen therefore that the array may be used to capture RNA, e.g. mRNA, of a biological specimen, e.g. tissue sample, that is contacted with said solid substrate, e.g. array. The methods disclosed herein may thus be considered as methods of quantifying the spatial expression of one or more transcripts in a tissue sample. Expressed another way, the methods disclosed herein may be used to detect the spatial expression of one or more transcripts in a biological specimen, e.g. tissue sample. In yet another way, the methods disclosed herein may be used to determine simultaneously the expression of one or more transcripts at one or more positions within a biological specimen, e.g. tissue sample. Still further, the methods may be seen as methods for partial or global transcriptome analysis of a biological specimen, e.g. tissue sample, with two-dimensional spatial resolution.

Embodiments

1. A solid substrate for performing spatially resolved transcriptome sequencing of one or more biological specimens, the solid substrate having a surface comprising:

an array of capture regions comprising substrate oligonucleotides bound to the solid substrate, the substrate oligonucleotides comprising a cleavable motif, a UMI, a first sector barcode, a second sector barcode, a first pixel barcode, a second pixel barcode, and a capture sequence, each capture region of the array being identifiable by the substrate oligonucleotide bound thereto;

wherein the first sector barcode is common to the substrate oligonucleotides of a series of adjacent rows of capture regions of the array, and the second sector barcode is a common to the substrate oligonucleotides of a series of adjacent columns of capture regions of the array, each combination of first sector barcode and second sector barcode defining a sector; and

each sector comprising multiple capture regions, each capture region comprising substrate oligonucleotides comprising a unique combination of first pixel barcode and second pixel barcode, wherein each unique combination of first pixel barcode and second pixel barcode defines x and y positions, respectively, within that sector.

2. A solid substrate for performing spatially resolved transcriptome sequencing of one or more biological specimens, the solid substrate having a surface comprising: an array of capture regions comprising substrate oligonucleotides bound to the solid substrate, the substrate oligonucleotides comprising a cleavable motif, a UMI, a first pixel barcode, a second pixel barcode, a first sector barcode, a second sector barcode, and a capture sequence, each capture region of the array being identifiable by the substrate oligonucleotide bound thereto;

wherein the first sector barcode is common to the substrate oligonucleotides of a series of adjacent rows of capture regions of the array, and the second sector barcode is a common to the substrate oligonucleotides of a series of adjacent columns of capture regions of the array, each combination of first sector barcode and second sector barcode defining a sector; and

each sector comprising multiple capture regions, each capture region comprising substrate oligonucleotides comprising a unique combination of first pixel barcode and second pixel barcode, wherein each unique combination of first pixel barcode and second pixel barcode defines x and y positions, respectively, within that sector.

3. The solid substrate of embodiment 1 or 2, wherein the array of capture regions comprises substrate oligonucleotides having a plurality of unique first sector barcodes and a plurality of unique second sector barcodes.

4. The solid substrate of any one of embodiments 1 to 3, wherein the array of capture regions comprises substrate oligonucleotides having at least four unique first sector barcodes and at least four unique second sector barcodes defining 16 sectors.

5. The solid substrate of any one of embodiments 1 to 4, wherein the array of capture regions comprises substrate oligonucleotides having a plurality of unique first pixel barcodes and a plurality of unique second pixel barcodes.

6. The solid substrate of any one of embodiments 1 to 5, wherein the array of capture regions comprises substrate oligonucleotides having about 96 unique first pixel barcodes spaced about 3 pm apart and about 96 unique second pixel barcodes spaced about 3 pm apart.

7. The solid substrate of any one of embodiments 1 to 6, wherein the capture regions are spaced about 3 pm apart to about 200pm apart in any direction. 8. The solid substrate of any one of embodiments 1 to 7, wherein the substrate oligonucleotides comprise linkers joining each of the first sector barcode, the second sector barcode, the first pixel barcode, and the second pixel barcode.

9. The solid substrate of any one of embodiments 1 to 8, wherein the substrate oligonucleotides further comprise a linker joining the second pixel barcode and the capture sequence.

10. The solid substrate of any one of embodiments 1 to 9, wherein the substrate oligonucleotides further comprise a primer handle.

11. The solid substrate of any one of embodiments 1 to 10, further comprising an offset UMI anchor.

12. The solid substrate of any one of embodiments 1 to 11, wherein the oligonucleotides further comprise a cleavable motif, optionally wherein the cleavable motif comprises i) a sequence of at least 3, 4, 5, or 6 deoxyuridine (dU) bases or ii) a restriction recognition site.

13. The solid substrate of any one of embodiments 1 to 12, wherein the capture sequence is a poly(d)T or poly(d)T VN.

14. The solid substrate of any one of embodiments 1 to 14, further comprising fiducial pixels comprising fiducial oligonucleotides bound to the solid substrate.

15. The solid substrate of any one of embodiments 1 to 14, wherein the fiducial pixels further comprise a fluorescent label.

16. The solid substrate of any one of embodiments 1 to 15, comprising 140,000 or more capture regions, optionally wherein the array of capture regions is an area of at least about 1 2

cm

17. The solid substrate of any one of embodiments 1 to 16, comprising an array of capture regions comprising up to about 9,200 unique capture regions in an area of about 9.2 mm² and/or up to about 1,800,000 unique capture regions in an area of about 8.0 mm².

18. The solid substrate of any one of embodiments 1 to 17, wherein the solid substrate is a functionalized glass slide, optionally a glass slide that is 75 x 25 x 1 mm. 19. The solid substrate of any one of embodiments 1 to 18, wherein the substrate oligonucleotides further comprise a UMI offset anchor, optionally wherein the UMI offset anchor is immediately 3’ to the UMI.

20. A method for generating a solid substrate having a surface comprising an array of capture regions, the capture regions being arranged in rows and columns, the method comprising:

i) contacting each row in a series of adjacent rows of a solid substrate with one of a set of first sector barcode oligonucleotides, each first sector barcode oligonucleotide comprising a first sector barcode and a first linker, wherein each first sector barcode oligonucleotide in the set has a different first sector barcode, wherein the first sector barcode oligonucleotides are bound to the solid substrate using a water-soluble amine linker, optionally bis(sulfosuccinimidyl)suberate (BS3), wherein the first sector barcode oligonucleotides comprise a 5’ amine modification;

ii) contacting each column in a series of adjacent columns of the solid substrate with one of a set of second sector barcode oligonucleotides, each second sector barcode oligonucleotide comprising a second sector barcode and a second linker, wherein each second sector barcode oligonucleotide in the set has a different second sector barcode, wherein each combination of first sector barcode and second sector barcode defines a sector,

iii) contacting each row in the series of adjacent rows of the solid substrate with a set of first pixel barcode oligonucleotides, each first pixel barcode oligonucleotide comprising a first pixel barcode and a third linker, wherein each first pixel barcode oligonucleotide in the set has a different first pixel barcode;

iv) contacting each column in the series of adjacent columns of the solid substrate with a set of second pixel barcode oligonucleotides, each second pixel barcode oligonucleotide comprising a second pixel barcode and a fourth linker, wherein each second pixel barcode oligonucleotide in the set has a different second pixel barcode, wherein each combination of first pixel barcode and second pixel barcode defines x and y positions, respectively, within a sector; and

v) contacting each column and row of the solid substrate with a capture oligonucleotide comprising a fourth linker and a capture sequence.

21. A method for generating a solid substrate having a surface comprising an array of capture regions, the capture regions being arranged in rows and columns, the method comprising: i) contacting each row in a series of adjacent rows of a solid substrate with one of a set of first sector barcode oligonucleotides, each first sector barcode oligonucleotide comprising a first sector barcode and a first linker, wherein each first sector barcode oligonucleotide in the set has a different first sector barcode, wherein the first sector barcode oligonucleotides are bound to the solid substrate having an amine-reactive functional groups, optionally N-oxysuccinimide, wherein the first sector barcode oligonucleotides comprise a 5’ amine modification;

ii) contacting each column in a series of adjacent columns of the solid substrate with one of a set of second sector barcode oligonucleotides, each second sector barcode oligonucleotide comprising a second sector barcode and a second linker, wherein each second sector barcode oligonucleotide in the set has a different second sector barcode, wherein each combination of first sector barcode and second sector barcode defines a sector,

iii) contacting each row in the series of adjacent rows of the solid substrate with a set of first pixel barcode oligonucleotides, each first pixel barcode oligonucleotide comprising a first pixel barcode and a third linker, wherein each first pixel barcode oligonucleotide in the set has a different first pixel barcode;

iv) contacting each column in the series of adjacent columns of the solid substrate with a set of second pixel barcode oligonucleotides, each second pixel barcode oligonucleotide comprising a second pixel barcode and a fourth linker, wherein each second pixel barcode oligonucleotide in the set has a different second pixel barcode, wherein each combination of first pixel barcode and second pixel barcode defines x and y positions, respectively, within a sector; and

v) contacting each column and row of the solid substrate with a capture oligonucleotide comprising a fourth linker and a capture sequence.

22. The method of embodiment 20 or 21, wherein the first sector barcode oligonucleotides comprise a cleavable motif, a UMI, and a first sector barcode, optionally further comprising a primer handle.

23. The method of any one of embodiments 20 to 22, wherein the cleavable motif cleavable motif comprises i) a sequence of at least 3, 4, 5, or 6 deoxyuridine (dU) bases or ii) a restriction recognition site.

24. The method of any one of embodiments 20 to 23, wherein, after step i), the first sector barcode oligonucleotide is bound to the substrate. 25. The method of any one of embodiments 20 to 24, wherein, after step i), the solid substrate is contacted with a set of first splint oligonucleotides, each first splint oligonucleotide in the set comprising a sequence complementary to the first linker and one of the second sector barcodes, whereby the first splint oligonucleotide is bound to the first sector oligonucleotide.

26. The method of any one of embodiments 20 to 25, wherein after step ii), the second sector barcode oligonucleotide is bound to the first splint oligonucleotide.

27. The method of any one of embodiments 20 to 26, wherein the first sector barcode oligonucleotides comprise a duplex region, the duplex region having an overhang having a sequence complementary to one of the second sector barcodes.

28. The method of any one of embodiments 20 to 27, wherein after step ii), the solid substrate is contacted with a set of second splint oligonucleotides, each second splint oligonucleotide in the set comprising a sequence complementary to the second linker and one of the first pixel barcodes whereby the second splint oligonucleotide is bound to the second sector oligonucleotide.

29. The method of any one of embodiments 20 to 28, wherein after step iii), the first pixel barcode oligonucleotide is bound to the second splint oligonucleotide.

30. The method of any one of embodiments 20 to 29, wherein the second sector barcode oligonucleotides comprise a duplex region, the duplex region having an overhang having a sequence complementary to one of the first pixel barcodes.

31. The method of any one of embodiments 20 to 30, wherein after step iv), the solid substrate is contacted with a set of third splint oligonucleotides, each third splint oligonucleotide in the set comprising a sequence complementary to the third linker and one of the second pixel barcodes whereby the third splint oligonucleotide is bound to the first pixel barcode oligonucleotide.

32. The method of any one of embodiments 20 to 31, wherein after step iv), the second pixel barcode oligonucleotide is bound to the third splint oligonucleotide. 33. The method of any one of embodiments 20 to 32, wherein the third sector barcode oligonucleotides comprise a duplex region, the duplex region having an overhang having a sequence complementary to one of the second pixel barcodes.

34. The method of any one of embodiments 20 to 33, wherein after step v), the solid substrate is contacted with a fourth splint oligonucleotide comprising a sequence complementary to the fourth linker and the capture oligonucleotide whereby the fourth splint oligonucleotide is bound to the second pixel barcode oligonucleotide.

35. The method of any one of embodiments 20 to 34, wherein after step v), the capture oligonucleotide is bound to the fourth splint oligonucleotide.

36. The method of any one of embodiments 20 to 35, wherein the fourth sector barcode oligonucleotides comprise a duplex region, the duplex region having an overhang having a sequence complementary to the capture sequence.

37. The method of any one of embodiments 20 to 36, wherein the capture sequence is a poly(d)T or poly(d)T VN.

38. The method of any one of embodiments 20 to 37, further comprising, after step v), performing a ligation reaction.

39. The method of any one of embodiments 20 to 38, further comprising performing a ligation reaction after step i), ii), iii), iv), and/or v).

40. The method of any one of embodiments 20 to 39, wherein ligation comprises addition of T4 ligase.

41. The method of any one of embodiments 20 to 40, further comprising removing the splint oligonucleotides.

42. The method of any one of embodiments 20 to 41, wherein in step i)

a) first sector barcode oligonucleotides are present at a concentration of about 0.1 pM to about 10 pM; and/or

b) the water-soluble amine linker is present at a concentration of about 2.5 mM.

43. A solid substrate obtained by the method of any one of embodiments 20 to 42. 44. A method of binding oligonucleotides to a solid substrate, the method comprising binding the oligonucleotides to the solid substrate using a water-soluble amine linker, optionally bis(sulfosuccinimidyl)suberate, wherein the oligonucleotides comprise a 5’ amine modification.

45. The method of embodiment 44, wherein

a) the oligonucleotides are present at a concentration of about 1 to about lOpM; and/or

b) the water-soluble amine linker is present at a concentration of about 2.5 mM.

46. A solid substrate obtained by the method of embodiment 44 or 45.

47. A method of spatially resolved transcriptome sequencing of one or more biological specimens, the method comprising:

a) providing the solid substrate of any one of embodiments 1 to 19 or 43;

b) mounting the one or more biological samples to the solid substrate, the biological samples partially or completely overlaying the array of capture regions;

c) performing staining or immunofluorescent labeling of the one or more biological samples;

d) capturing one or more images of the one or more biological samples;

e) permeabilizing the one or more biological specimens thereby permitting RNA present in the one or more biological specimens to bind capture sequences of the substrate oligonucleotides;

f) performing reverse-transcription by addition of a reverse transcriptase under conditions suitable to permit generation of cDNA:RNA hybrid molecules, wherein the reverse transcription includes extension of RNA using the bound substrate oligonucleotide as a template to result in tagging of the RNA with sequences complementary to the first sector barcode, the second sector barcode, the first pixel barcode, and the second pixel barcode of the bound substrate oligonucleotide; and

g) sequencing tagged RNA and assigning sequences to a capture region.

48. The method of embodiment 47, wherein the solid substrate is contacted with fluorescently tagged oligonucleotide that hybridize with the fiducial pixels.

49. The method of embodiment 47 or 48, wherein the reverse transcriptase is a hybrid reverse transcriptase-helicase enzyme. 50. The method of any one of embodiments 47 to 49, wherein the reverse transcriptase is a modified MMLV reverse transcriptase (RT), Maxima H Minus Reverse Transcriptase, SuperScript™ II Reverse Transcriptase, Induro® Reverse Transcriptase, M-MLV Reverse Transcriptase, UltraMarathonRT (RNA Connect).

51. The method of any one of embodiments 47 to 50, further comprising releasing the substrate oligonucleotides bound to the solid substrate by addition of an enzyme that cleaves the cleavable motif of the oligonucleotides.

52. The method of any one of embodiments 49 to 51, wherein the enzyme that cleaves the cleavable motif is added following step f).

53. The method of any one of embodiments 49 to 52, wherein step f) further comprises addition of an enzyme that cleaves the cleavable motif of the oligonucleotides.

54. The method of any one of embodiments 49 to 53, further comprising adapter ligation, wherein a ligase enzyme covalently attaches a sequencing adapter to the RNA of the cDNA:RNA hybrid molecules, and performing sequencing of the RNA, optionally wherein the sequencing is long run sequencing and/or nanopore sequencing.

55. The method of any one of embodiments 49 to 54, wherein the capture sequences are poly(d)T or poly(d)T VN.

56. The method of any one of embodiments 44 to 54, wherein up to 16 biological samples are mounted to the array, each sample being mounted to a sector of the array identifiable by a combination of the first sector barcode and the second sector barcode.

57. The method of any one of embodiments 49 to 56, wherein the method does not comprise second strand cDNA synthesis or PCR amplification.

EXAMPLES

The following examples are provided for purposes of illustration only. The protocols and methods described in the examples are not considered to be limitations on the scope of the claimed invention. Rather this specification should be construed to encompass any and all variations that become evident as a result of the teaching provided herein. One of skill in the art will understand that changes or variations can be made in the disclosed embodiments of the examples and expected similar results can be obtained. For example, the substitutions of reagents that are chemically or physiologically related for the reagents described herein are anticipated to produce the same or similar results. All such similar substitutes and modifications are apparent to those skilled in the art and fall within the scope of the invention.

Example 1: miST Platform

The micro isoform ST (miST) is a novel method that combines long read sequencing with spatially resolved sequencing (FIGs. 1A-1C). FIGs. lAto 1C show an overview of combinatorial microfluidics devices used for the fabrication of (FIG. 1 A) sixteen 96-by-96 channel subarrays via (FIG. IB) duplex ligation or (FIG. 1C) hybridization-and-extension DNA polymerization. Density ranges may be varied, e.g., (a) lowest density: 96 x 96 x 100 pm devices x 1 sector = -9,200 unique pixel barcodes in about 9.2 mm² area, and (b) highest density: 96 x 96 x 3 pm devices x 14 x 14 sectors = ~1.8million unique pixel barcodes in about 8 mm² area.

For surface functionalization process, in miST platform, production of high-density spatial capture surfaces is performed and achieved via amine-based covalent coupling chemistry. Previous low-density surfaces used in commercial platforms such as Visium from 10X Genomics used low quality substrates for aminated DNA oligo deposition. The quality of the capture surface and the density of the aminated DNA deposition is proportional to efficiency of capture of substrates from cell and tissues onto the spatially barcoded surface. We have identified concentrations of DNA oligonucleotides used to successfully attach the DNA barcodes to the glass substrate (FIG. 2). FIG. 2 shows FISH (fluorescent in situ hybridization) images (intensities of the white signal indicates poly(d) T presence) of optimizing DNA to reducing agent ratios landing at a 1-10 pM DNA concentration used in our formulations.

In miST platform we introduced a precise sector barcoding schema. One major hurdle in spatial technology is capture surface production. The number of capture spots (or squares) is directly proportional to the number of oligonucleotides that can be patterned on a glass surface. Current commercialized technologies (such as DBIT-seq, AtlasXOmics) can only barcode 1mm² of a glass slide and a tissue section, which technologies use a process termed deterministic barcoding, where through two orthogonal PDMS (polydimethylsiloxane) devices (channel width minimum 10 pm), they introduce 100 x 100 with 10,000 different barcoded spots. This is insufficient as most tissue blocks are more than 1 cm² in size. In order to be able to produce a large surface area but still use only 100 x 100 DNA oligo design and principle, we introduced an additional set of PDMS devices to introduce sector barcodes. In recent papers, such as MAGIC-Seq, the authors also introduced a concept of sector (e.g., Zhu, J., Pang, K., Hu, B. et al. Custom microfluidic chip design enables cost-effective three-dimensional spatiotemporal transcriptomics with a wide field of view. Nat Genet (2024)). However, MAGIC-Seq technology is based on manual deposition of sector barcodes with no avenue to perform image registration and alignment. In miST platform, our precise sector barcoding schema (e.g., 4 x 4 wide channel 1 mm devices) can pre-barcode a capture area very accurately (FIG. 3), which also enables accurate image registration in the next steps. FIG. 3 shows FISH images of poly(d)T -stained sector arrays using competing approaches for sector barcode deposition (left) and mi ST (right).

Additionally, we designed specific 100 x 100 channel PDMS (pixel devices) devices to flow barcodes over the pre-barcoded sector area to cheaply and robustly enable spatial barcoding of large tissue areas. Combinatorial sector and pixel barcoding, through the use of microfluidic devices, enables inexpensive and scalable synthesis of high-resolution, spatially barcoded, oligonucleotide microarrays (FIG. 4). FIG. 4A and FIG. 4B show FISH stains of mi ST arrays production for a 16-sector device. FIG. 4 A shows Each individual yellow square represents a single 10pm pixel that has a unique spatial barcode. FIG. 4B shows zoom in on an array representing one of the sixteen sectors (cyan inset in FIG. 4A). Color code: The poly(dT) capture sequence, deposited across rows with a pixel device, is labeled in yellow. The linker sequence, deposited across columns using a pixel device, is shown in red. By our innovative PDMS microfluidics design, we also made our technology easily compatible with regular size glass slides (75 x 25 x 1 mm) (as compared to MAGIC-seq). This is of value to the users given most microscopes can only operate on regular glass slides given the device holder dimensions and which will make it easily adaptable for wide use. The design of our spatial capture surface is modular, and can be run as a single capture device or up to 16 capture devices in parallel.

Additionally, in miST platform, we present a new formulation of the chemistry using water-soluble amine linkers (e.g., bis(sulfosuccinimidyl)suberate) (BS3) to conjugate nucleic acids to a functionalized glass surface to generate a capture surface of even density (FIG. 5). FIG. 5 A and FIG. 5B show immunofluorescence stains of miST arrays that were produced using two different formulations. FIG. 5 A shows single-sector array fabricated disuccinimidyl suberate-linked dendrimer surface (left) bis(sulfosuccinimidyl)suberate-linked dendrimer surface (right) and with 10 gm pixels. FIG.5B shows zoomed image of array insets (white rectangles in FIG. 5A) synthesized using either disuccinimidyl suberate (left) or bis(sulfosuccinimidyl)suberate (right). The poly(dT) capture sequence, deposited across rows with a pixel device, is labeled in yellow. The fiducial sequence, found only in the columns of pixels flanking the array, are labeled in red. Our formulation is: 2.5 mM BS3, 1-10 pM DNA oligo and IX phosphate buffered saline (PBS) incubate at 25°C for 1 hour for efficient DNA oligonucleotide conjugation. The surface area is then washed with 2X Saline-sodium citrate (SSC) twice, followed by a wash 2X SSC + 0.1% SDS (sodium dodecyl sulfate) and followed by three washes with miliQ water. The slides are then blocked at room temperature for 40 minutes with 50 mL of blocking mix (8 g succinic anhydride, ~49.5ml dimethyl formamide, and 500 pL triethylamine. Slides are then finally rinsed 3 times with ~40ml miliQ water and spun dried.

Additionally, in miST platform we introduce fiducial pixels labeled with fluorescent oligonucleotides (FIG. 6). FIG. 6A and FIG. 6B show FISH visualization of the pixel frame probe. FIG. 6A shows a single sector array visualized on the glass surface using the complementary frame probe (red) and poly(d)T yellow. FIG. 6B shows a zoom in (inset from FIG. 6A) on frame probe and showing example image processing for frame probe detection. White squares show the frame probe pixels on the spatial array. These fiducial pixels enable accurate registration of all produced pixel channels simultaneously or in combination with tissue staining when placed on the spatial capture surface. This enables a very fast quality control of the produced spatial array as well as very fast concatenation of spatial barcodes within or outside the sampled tissue area (FIG. 7). FIG. 7 shows array and tissue registration visualization. A DAPI stained mouse brain tissue on top of a miST spatial array (left) and zoom in (insets from right).

In order to make miST platform fully compatible with long read sequencing, we ensured that all of the sector and pixel barcodes are designed in a combinatorial, error-robust way. Our barcode design ensures that the barcodes do not contain any DNA base repeats or homopolymeric regions, that they all have 50% GC content, and that the linker sequences needs to the combinatorial sector or pixel orthogonal barcoding end with degenerate nucleotides such that any barcode sequence flanking the linker will begin or end with a different nucleotide, thus removing the possibility of a DNA base repeat (FIGs. 8-9). FIG. 8 shows example designs of the first 4 sector barcodes used in the first PDMS device that attaches oligos to the glass surface. The design indicate the attachment chemistry (5'-Amine modification followed by a C6 linker), a stretch of dU nucleotides, an optional PCR primer handle that is adaptable with multiple sequencing technologies and library preparation strategies (and is fully interchangeable), a degenerate UMI sequence where V and D are degenerate bases to determine the start and stop of the UMI sequence, Offset UMI-Anchor, a sector barcode (where the 4 different sequences exemplify the different sector barcodes) followed by a Linker A sequence, which is used in the next PDMS device to hybridize an orthogonal set of sector barcodes through a Linker-A’ sequence. FIG. 9 shows final barcode design metrics. Each of the pixel barcodes is 10 nucleotides long, has 50% GC content, there are no homopolymer repeats, we minimized the shared k-mer length and maximized the pairwise distance (tested for Levenshtein and Hamming).

Further, we designed mi ST platform in a way that the sector and pixel barcodes are combinatorially assembled onto the glass surface via two types of enzymatic reactions: ligation and hybridization-and-extension via Klenow. The Klenow reaction had been previously described in Lbtstedt et al (Lbtstedt, B., Strazar, M., Xavier, R. et al. Spatial hostmicrobiome sequencing reveals niches in the mouse gut. Nat Biotechnol 42, 1394-1403 (2024)), while we have improved the ligation reaction. The formulation for the ligation reaction is: IX T4 ligase buffer loaded with 22,500 units of T4 ligase. We show that the new ligation chemistry greatly increases the percentage of barcodes that can be called using standard bioinformatics pipeline (FIG. 10). FIG. 10 provides a table showing comparison of sequencing accuracy in combinatorial barcodes synthesized by either hybridization-and-extension (“Klenow”) or ligation. Values shown are the percent of reads which have a flanking DNA adapter sequence detected with either a perfect match to the whitelisted barcode sequences and the percent of reads with fewer than three mismatches (“Hamming Distance < 3”).

Further, to increase the transcript detection efficiency (defined as the number of unique molecules per gene that are detected in each spatial barcode), first, we improved the reverse transcription (RT) reaction conditions of the miST array surface (FIG. 11). FIG. 11 shows immunofluorescence images of spatial gene activity of an adult mouse brain. Intensity of the white signal indicates more or longer mRNA molecules have been spatially transcribed in the cDNA synthesis steps using Decoder-seq cDNA RT conditions (left) and miST conditions (right) on consecutive mouse brain sections. Stronger signal intensities are favorable as they indicate more cDNA product has been transcribed. The RT reaction is loaded with IX RT buffer, 0.2-0.4 mg BSA, 0.5-1 pmol dNTPs, 5-15 nmol template switch oligo, 0.1X PEG 8000 (50% w/v), 8 pl RT Maxima H- enzyme and 4 pl RNAOUT (Invitrogen) inhibitor. The reaction volumes were 80 pl. This reaction was run first with the same RT enzyme as Decoder-seq (Cao, J., Zheng, Z., Sun, D. et al. Decoder-seq enhances mRNA capture efficiency in spatial RNA sequencing. Nat Biotechnol (2024)) for comparisons. Our improved reaction conditions were run overnight at 42°C for optimal results. This reaction optimization is also compatible with other spatial technologies (such as Visium, Curio, or DBIT-seq (Liu, Y., et al., High-Spatial-Resolution Multi-Omics Sequencing via Deterministic Barcoding in Tissue, Cell, 2020, 183(6):P 1665- 1681 , el8)). Second, we performed a release of the spatial barcodes from the glass surface into the tissue, which has an effect on improving RT efficiency (Vickovic, S., Lbtstedt, B., Klughammer, J. et al. SM-Omics is an automated platform for high-throughput spatial multi-omics. Nat Commun 13, 795 (2022)). We further tested and confirmed that the probe release from our new spatial surface is possible using the aforementioned reaction conditions (FIG. 12). FIG.

12 shows immunofluorescence images (intensities of the white signal indicate poly(d)T presence) of optimizing cDNA:mRNA hybrid cleavage reactions from the spatial surface. “Cleavage reaction ON” indicates the cleavage reaction was applied to replicate spatial arrays (columns) while “Cleavage reaction OFF” indicates the conditions where no cleavage enzyme was added to the reaction. Decrease in while signal intensities in the “Cleavage reactions ON” condition” as compared to the “Cleavage OFF” condition indicated successful cleavage of the cDNA:mRNA hybrid.

Further in miST platform, we also present a discovery we made using this chemistry that was previously unknown. We can directly spatially barcode RNA molecules through DNA-templated extension with reverse transcriptase enzymes, which is an extremely important step that is needed to enable spatial long read sequencing (FIG. 13). FIG. 13 shows a workflow for (i) direct RNA capture, (ii) barcoding via reverse transcription, (iii) enzymatic release from slide, (iv) adapter ligation, and (v) nanopore sequencing. We found that several reverse transcriptase enzymes retain this activity, however only enzymes with no RNase activity are capable of barcoding full length RNA molecules. During this step, we also used RT enzymes that are more processive than the Maxima H- previously used by us and others (FIG. 14). FIG. 14A and FIG. 14B show barcode transfer via DNA-templated extension with reverse transcriptase enzymes. FIG. 14A shows RNA integrity after reverse transcription and first strand cDNA degradation with DNase. The x-axis shows the length of the RNA molecules and the y-axis shows the relative abundance of molecules of a particular length. FIG. 14B shows PCR amplification of a barcoded RNA library using either a full barcode and poly(dT)VN primer or just the barcode sequence. The x-axis shows the length of the cDNA molecules and the y-axis shows the relative abundance of molecules of a particular length.

Finally, to improve the sensitivity of spatial long read measurements, we identified chemical conditions needed to apply processive reverse transcriptase enzymes to glass surfaces (FIG. 15). FIG. 15A - FIG. 15D shows conditions for surface capture and release of full-length RNA-DNA hybrid molecules. Length analysis of DNA/RNA hybrid molecules after surface capture, reverse transcription, and enzymatic release when (FIG. 15 A) no enzyme was included in the reverse transcription step, (FIG. 15B) Maxima H-Minus RT was included using previously demonstrated conditions, (FIG. 15C) the commercially recommended concentration of Ultra Marathon RT was included in the reverse transcription step, or (FIG. 15D) Ultra Marathon RT was used at an higher concentration in the reverse transcription step. Unlike existing methodologies, here we demonstrate the ability to capture, barcode, and release full length RNA molecules on functionalized glass surfaces. This novel chemistry enables sequencing to be performed directly on barcoded RNA molecules, without the need for second strand cDNA synthesis or PCR amplification.

Example 2: miST - Materials and Methods

Described below is an exemplary protocol for long read library preparation.

Oligos

TSO 5’-AAGCAGTGGTATCAACGCAGAGTACATrGrGrG-3’ (SEQ ID NO: 201)

uMRT-TSO 5’-CCCTCTCTCTCTCTTTCCTCTCTCTTTT-3’(SEQ ID NO: 202) Full-lOxTSO 5’-AAGCAGTGGTATCAACGCAGAGTACATGGG-3’(SEQ ID NO: 203)

Partial- lOxTSO 5’-AAGCAGTGGTATCAACGCAGAG-3’(SEQ ID NO: 204) uMRT-AmpPCR 5’-CCCTCTCTCTCTCTTTCCTCTCTC-3’(SEQ ID NO: 205) lOxcDNA 5’-CTACACGACGCTCTTCCGATCT-3’(SEQ ID NO: 206)

5 ’-Biotinylated 1 OX-Full-cDNA /5Biosg/- CAGCACTTGCCTGTCGCTCTATCTTC CTACACGACGCTCTTCCGATCT-3 ’(SEQ ID NO: 207)

Rev_PR2_partial_TSO_defined 5’-CAGCTTTCTGTTGGTGCTGATATTGC AAGCAGTGGTATCAACGCAGAG-3’(SEQ ID NO: 208)

anti-fiducial frame 5’-GGTACAGAAGCGCGATAGCAG-3’ (SEQ ID NO: 209) (can be 5 ’FAM, 5’CY5 or 5’CY3)

Fiducial probe staining prior to sectioning (optionally performed)

1. Dilute anti-fiducial frame probe freshly to 0.1-1 pM in lx PBS.

2. Add to miST prior to Tissue sectioning.

3. Incubate for 10-30min at room temperature.

4. Pipette out the solution and wash the slide by dipping in a 50 mL Falcon tube prefilled with IX PBS.

5. Dry slide using a slide spinner.

6. If this step was performed, co-staining with DAPI can be skipped in the DAPI and Fiducial probe staining section.

Tissue sectioning

Slide needs to be equilibrated to -20°C for at least 20 minutes (min) before placing a tissue section on top. Cryosection fresh frozen tissue section on a pre-chilled miST slide. Thickness of section can vary from 5 pm to 25 pm. Once ready to start a miST experiment, warm miST slide 1 minute 37°C. Proceed immediately to tissue fixation.

Tissue fixation

Prechill 50mL falcon tube filled with Methanol to -20°C. Place slide with tissue in ice cold Methanol and incubate at -20°C for 30 minutes. Take slide out of falcon tube pipette around 500 pl isopropanol on top of the tissue. Let air dry for max lOmin. Remove excess isopropanol if any if left on slide with a Kimwipe. Proceed to DAPI staining.

DAPI and fiducial probe staining

Dilute DAPI freshly 1 : 1000 in lx PBS. Dilute anti-fiducial frame probe freshly to 0.1-1 pM in lx PBS DAPI solution. Pipette ~75-200pl of the above solution per well with tissues. Incubate 5 minutes atRT. Pipette out DAPI. Wash 3x 100-200 pl IxPBS. Take slide out of the slide holder. Dip the slide in lx PBS in a Falcon tube. Tip the slide vertically. Let air dry for max 10 minutes. Remove excess PBS if any if left on slide with a Kimwipe. Image using DAPI and anti-fiducial channel (determined by probe synthesis; either FAM, CY3 or CY5)

H&E tissue staining (alternative H&E tissue staining may be performed after the RT reaction)

Before starting the experiment: Dispense the following volumes of Milli-Q water: 50 ml in one 50-ml centrifuge tube/slide, 800 ml in Beaker 1, 800 ml in Beaker 2, 800 ml in Beaker 3.

Prepare Tris-Acetic Acid Buffer (0.45M, pH 6.0): prepare 200 ml (200 slides), store at room temperature, dissolve 11 g Tris base in 100 ml nuclease-free water, adjust pH to 6.0 using 100% Acetic Acid, bring volume to 200 ml with nuclease-free water, filter through 0.2 pM Coming 250 ml Vacuum Bottle.

Prepare Eosin mix (1 ml/slide): mix 100 pl of Eosin Y solution with 900 pl of Tris buffer (0.45 M, pH 6.)

Procedure:

1. Remove slide from methanol and wipe excess liquid from the back of the slide, without touching the tissue sections. Place on a flat, clean, nonabsorbent work surface.

2. Add 500 pl isopropanol to uniformly cover all tissue sections on the slide.

3. Incubate 1 minute at room temperature.

4. Discard reagent by draining and/or holding the slide at an angle with the bottom edge in contact with a laboratory wipe.

5. Wipe excess liquid from the back of the slide, without touching the tissue sections. Place on a flat, clean, nonabsorbent work surface.

6. Air dry the slide.

7. Add 1 ml Hematoxylin to uniformly cover all tissue sections on the slide.

8. Incubate 7 minutes at room temperature.

9. Discard reagent by draining and/or holding the slide at an angle with the bottom edge in contact with a laboratory wipe.

10. Immerse the slide 5x in the water in 50 ml tube.

11. Immerse the slide 15x in the water in Beaker 1.

12. Immerse the slide 15x in the water in Beaker 2.

13. Wipe excess liquid from the back of the slide without touching the tissue section. Place on a flat, clean, nonabsorbent work surface. Some droplets may remain 14. Add 1 ml Bluing Buffer to uniformly cover all tissue sections.

15. Incubate 2 minutes at room temperature.

16. Discard reagent by draining and/or holding the slide at an angle with the bottom edge in contact with a laboratory wipe.

17. Immerse the slide 5x in the water in Beaker 2.

18. Wipe excess liquid from the back of the slide without touching the tissue section. Place on a flat, clean, nonabsorbent work surface. Some droplets may remain.

19. Add 1 ml Eosin Mix (1:10 dilution) to uniformly cover all tissue sections. Incubation time needs to be optimized on a per tissue base (e.g., 10 - 90 seconds interval).

20. Incubate 1 minute at room temperature.

21. Discard reagent by draining and/or holding the slide at an angle with the bottom edge in contact with a laboratory wipe.

22. Immerse the slide 15x in the water in Beaker 3. (The number of washes can vary between 5 and 15; the goal is to have a bright staining without a specific binding of the dye).

23. Wipe the back of the slide with a laboratory wipe. Place on a flat, clean, nonabsorbent work surface and air dry until tissue is opaque. Alternatively, you can use a microcentrifuge with slide adaptors to remove excess liquid by centrifugation.

24. Incubate slide on the Thermocycler Adaptor with the thermal cycler lid open for 5 minutes at 37°C.

Pepsin Permeabilization

1. Bring one tube 0.1% pepsin/0.1 M HC1 from the freezer and pre-heat to 37°C when during previous step. One tube should contain 400 pl, enough volume for 6 wells.

2. Take the slide, still attached to the Arraylt incubation chamber after the DAPI treatment & wash and set a pipette to 75 pl.

3. Add around 70 pl of the 75 pl 0.1% pepsin/0.1 M HC1 to each well; make sure you do not induce any bubbles in the wells.

4. Attach a plastic sealer and incubate in an thermomixer at 37°C for 30 seconds - 30 minutes.

5. Remove the pepsin/HCl from the wells by slow pipetting.

6. Wash each well by adding 100-200 pl O.lxSSC and then removing it slowly. 7. Proceed immediately with cDNA synthesis.

cDNA synthesis

cDNA synthesis with MmulV

1. Prepare a reverse transcription (RT) mix. The below calculation is enough for 1 well (adjust accordingly):

a. lOx RT MMulV buffer (NEB) 5.7 pl

b. water 53.3 pl

c. dNTP mix (lOmM each) 4 pl 2. When ~3 min of the pepsin incubation remain, add the following reagents:

a. RNase Out 2 pl

b. MMulV enzyme (NEB) 7 pl

3. Take the slide, still attached to the Array It incubation chamber after the pepsin treatment and set a pipette to 75 pl.

4. Add around 70 pl of the 75 pl RT mix to each well, make sure you do not induce any bubbles in the wells.

5. Cover wells with a plate sealer.

6. Incubate at 37-42°C for 30 minutes to 20 hours.

7. Remove the RT mixture.

8. Wash each well with 100 pl 0.1 x SSC.

cDNA synthesis with Maxima H Minus

1. Prepare a reverse transcription (RT) mix. The below calculation is enough for 1 well (adjust accordingly):

a. 5x RT Maxima H minus buffer (NEB) 15 pl b. water 45.75 pl

c. dNTP mix (lOmM each) 4 pl

d. TSO (100 uM) 5.6 pl

2. When ~3 min of the pepsin incubation remain, add the following reagents:

a. RNase Out 2 pl

b. Maxima H minus enzyme (NEB) 3.75 pl

3. Take the slide, still attached to the Array It incubation chamber after the pepsin treatment and set a pipette to 75 pl.

4. Add around 70 pl of the 75 pl RT mix to each well, make sure you do not induce any bubbles in the wells.

5. Cover wells with a plate sealer.

6. Incubate at 37-50°C for minimum 30 minutes - 20 hours.

7. Remove the RT mixture.

8. Wash each well with 100 pl 0.1 x SSC.

cDNA synthesis with UltraMarathon (low cone.)

1. Prepare a reverse transcription (RT) mix. The below calculation is enough for 1 well (adjust accordingly):

a. 2x Ultramarathon buffer (RNAConnect) 37.5 pl b. 20x Ultramarathon Boost (RNAConnect). 3.75 pl

c. water 18.4 pl

d. dNTP mix (lOmM each) 4 pl

e. TSO (100 pM) 5.6 pl

2. When ~3 min of the pepsin incubation remains, add the following reagents:

a. RNase Out 2 pl

b. Ultramarathon enzyme (RNAConnect) 3.75 pl

3. Take the slide, still attached to the Array It incubation chamber after the pepsin treatment and set a pipette to 75 pl.

4. Add around 70pl of the 75 pl RT mix to each well, make sure you do not induce any bubbles in the wells.

5. Cover wells with a plate sealer.

6. Incubate at either room temperature or 42°C min for a minimum of 30 minutes and up to 20 hours (no shake).

7. Remove the RT mixture.

8. Wash each well with lOOpl 0.1 x SSC.

cDNA synthesis with UltraMarathon (high cone)

1. Prepare a reverse transcription (RT) mix. The below calculation is enough for 1 well (adjust accordingly):

a. 2x Ultramarathon buffer (RNAConnect) 37.5 pl b. 20x Ultramarathon Boost (RNAConnect). 3.75 pl

c. water 14.65 pl

d. dNTP mix (lOmM each) 4 pl

e. TSO (100 pM) 5.6 pl

2. When ~3 min of the pepsin incubation remains, add the following reagents:

a. RNase Out 2 pl

b. Ultramarathon enzyme (RNAConnect) 7.5 pl

3. Take the slide, still attached to the Array It incubation chamber after the pepsin treatment and set a pipette to 75 pl.

4. Add around 70pl of the 75 pl RT mix to each well, make sure you do not induce any bubbles in the wells.

5. Cover wells with a plate sealer.

6. Incubate at either room temperature or 42°C min for a minimum of 30 minutes and up to 20 hours (no shake).

7. Perform Template Switching reaction with Ultramarathon

a. Add and equal volume of the TS mixture to the RT reaction: 5x template switching buffer 30 pL, UltraMarathonRT (20 units/pL) 15 pL, uMRT-TSO (10 pM) 15 pL, dATP (10 mM) (e.g., NEB, Cat# N0440S) 15 pL.

9. Wash each well with lOOpl 0.1 x SSC.

cDNA synthesis with Induro

1. Prepare a reverse transcription (RT) mix. The below calculation is enough for 1 well (adjust accordingly):

a. 5x Induro buffer (NEB). 15 pl

b. water 33.6 pl

c. dNTP mix (lOmM each) 4 pl

2. When ~3 min of the pepsin incubation remain, add the following reagents:

a. RNase Out 2 pl

b. Induro enzyme (NEB) 3.75 pl

3. Take the slide, still attached to the Array It incubation chamber after the pepsin treatment and set a pipette to 75 pl.

4. Add around 70pl of the 75 pl RT mix to each well, make sure you do not induce any bubbles in the wells.

5. Cover wells with a plate sealer.

6. Incubate at 37-55°C for 30 minutes to 20 hours (no shake).

7. Remove the RT mixture.

8. Wash each well with lOOpl 0.1 x SSC.

9. Leave the mask on as the next step is tissue removal.

Note: All of the above RT reactions can be supplemented with 4-12 pl PEG 8000 (50%). All of the above reactions can be supplemented with 2-8 pl of the USER enzyme (1000 U / mL, NEB). All of the above reactions can use alternative RNase Inhibitors, either enzymatic or synthetic, (at the same final w/v) such as SUPERaselN (Ambion), RNase Inhibitor, Murine (NEB) or Sequema. RNAse inhibitors can be added at w/v 2-8 pl. These reactions can be supplemented with ~0.2-0.4mg/ml BSA. In the above reactions, dNTPs can vary at 0.5-0.8 mM final concentration. In the above reactions, TSO can vary 6-9pM final concentration. Release after cDNA synthesis

Tissue removal

1. Prepare a proteinase K mix. The below calculation is enough for 1 well (adjust accordingly):

a. prK buffer (Qiagen or NEB) 65.3 pl

b. prK enzyme (Qiagen or NEB) 9.7 pl

2. Take the slide, still attached to the Arraylt incubation chamber after the pepsin treatment and set a pipette to 75 pl.

3. Add around 70pl of the 75 pl tissue removal mix to each well, make sure you do not induce any bubbles in the wells.

4. Cover wells with a plate sealer.

5. Incubate at 37-56°C for 30 minutes to 20 hours (no shake).

6. Remove the tissue removal mixture.

7. Wash each well with lOOpl 0.1 x SSC.

Cleavage of products from the surface

1. Prepare a cleavage mix. The below calculation is enough for 1 well (adjust accordingly):

a. lOx CutSmart Buffer (NEB) 7.5 pl

b. water 60 pl

c. USER enyme (1000 U/mL, NEB) 7.5 pl

2. Take the slide, still attached to the Arraylt incubation chamber after the pepsin treatment and set a pipette to 75 pl.

3. Add around 70 pl of the 75 pl cleavage mix to each well, make sure you do not induce any bubbles in the wells.

4. Cover wells with a plate sealer.

5. Incubate at 37-55°C for 30 minutes to 20 hours (no shake).

6. Remove the cleavage mixture. This is now your material to be used in the next steps.

If release (as described above) was not performed during the RT or release steps, the following steps need to be taken in order to collect the material from the miST surface:

Denaturation:

1. Remove the RT mixture.

2. Add 70pl 0.08 M KOH (freshly diluted from stock) to each well 3. Incubate 5 minutes at room temperature

4. Using a pipette, remove KOH from the wells.

5. Add lOOpl EB (10 mM Tris-Cl, pH 8.5) to each well.

TSO primer annealing

1. Prepare a primer hybridization. The below calculation is enough for 1 well (adjust accordingly):

a. 2x primer hybridization buffer 40 pl

b. Primer (I M) (P-lOx-TSO) 1.6 pl

c. H₂O 38.4 pl

2. Add 70pl primer annealing mix to each well; incubate at room temperature for 30 minutes.

3. Pipette out primer annealing mix

4. Wash with EB (10 mM Tris-Cl, pH 8.5) by pipetting lOOpl in each well and immediately pipette it off.

Q5 second strand synthesis

1. Prepare second strand mix. Keep mix on ice until in use. The below calculation is enough for 1 well (adjust accordingly):

a. 2nd strand primer (P-lOxTSO) (AAGCAGTGGTATCAACGCAGAGTACATGGG) (SEQ ID NO: 210)

(lOmM) 8 pl

b. 2X Q5 High Start Fidelity master mix (non hot start) 40 pl c. Water 32 pl

2. Add 70pl second strand mix to each well.

3. Apply slide seal on clamp and place on Eppendorf thermocycler. Incubate at 65°C for 20 minutes - 1 hour. Ramp down to 37°C. When chamber is at 37°C, it is safe to proceed to next step.

Denaturation

1. Remove reagents from the wells.

2. Add lOOpl Buffer EB to each well and remove Buffer EB immediately from the wells.

3. Add 35pl 0.08 M KOH (diluted from stock) to each well. Add plastic seal.

4. Incubate 10 minutes at room temperature with 300-rpm shaking.

5. Prepare 5 pl Tris (1 M, pH 7.0) to up to an 8-tube strip. 6. Transfer 35 pl sample from each well to an individual tube containing Tris in the 8-tube strip.

7. Vortex, centrifuge briefly, and place on ice. Proceed immediately to next step.

In case the denaturation step was performed in larger volumes, use the Oligo Clean and Concentrate kits from Zymo Research. Example protocol when eluting in 200 pl below:

1. Add 400 pl Oligo Binding Buffer to 200 pl sample.

2. Add 800 pl or EtOH (95-100%) and mix well by pipetting.

3. Transfer the sample to the Zymo-Spin™ IC Column in a Collection Tube and centrifuge. Discard the flow-through. It will take 3x to load this much volume so repeat step 3 several times (e.g., 600 pl, 600 and then 200 pl loads).

4. Add 750 pl DNA Wash Buffer to the column and centrifuge for 1 minute ensure complete removal of the wash buffer. Carefully, transfer the column into a nuclease-free tube (not provided).

5. Do a serial dilution from the columns (10 pl, 10 pl and then 20 pl). Add first load directly to the column matrix and centrifuge. Repeat for the other two loads. Do not change collection tubes.

Pre-amplify the library

1. Prepare the below mix on ice:

a. Sample (from denaturation step). 40 pl

b. 10X cDNA oligo or uMRT-AmpPCR oligo (10 pM) 5 pl

c. Partial- 1 OX-TSO oligo or Full- 1 OX-TSO oligo (10 pM) 5 pl d. 2X NEB Next HiFi PCR mix or lx KAPA HiFi PCR Mix 50 pl

2. Run PCR for 5-20 cycles using the following protocol: 98°C 3 minute for 1 cycle, 98°C 15 seconds for 5-20 Cycles, 67°C 20 seconds, 72°C 1 minute, 72°C 1 minute 1 cycle, 4°C hold.

Note: In case uMRT primers are used, add 10 pl of uMRT-AmpPCR oligo, sample and the PCR mix. In case of 10X Oligos, use a combination of lOXcDNAoligo (5 pl) and either Partial- 1 OX-TSO oligo or Full- 1 OX-TSO oligo (5 pl).

Beads purification (0.6X)

1. Add 60 pl beads to each of well. 2. Mix 100 pl sample with 60 pl beads by pipetting 10 times, bind for 15 minutes. 3. Separate beads at magnetic rack for 5 minutes.

4. Remove buffer by slow pipetting; make sure not to touch the beads.

5. Add lOOOpl 80% ethanol (EtOH) to each tube. After about 30 seconds, slowly remove the EtOH by pipetting. Make sure not to touch the beads.

6. Repeat previous step two more times for a total of three times.

7. Let air dry, with the lids open, until you see the first signs of cracking (10-15 minutes).

8. Suspend beads in 25 pl water (RNase/DNase free).

9. Incubate for 2 minutes (not on magnetic rack).

10. Place tubes on magnetic rack for 5 minutes.

11. Collect 23 pl from each tube on the magnetic rack.

12. Store material in -20°C.

Amplify the library with Biotin TouchDown PCR

1. Prepare the below mix on ice:

a. Sample (from clean up step) 21 pl

b. 5 ’-Biotinylated lOX-Full-cDNA or uMRT-AmpPCR oligo (10 pM) 2 pl c. Rev_PR2_partial_TSO_defined (10 pM) 2 pl

d. 2X LongAmp Taq mix (NEB) 25 pl

2. Run PCR for 4 cycles (4 cycles preferred, only increase cycle count on >2nd PCR attempts) using the following protocol: 94°C 30 seconds 1 cycle, 94°C 15 seconds for 4-6* Cycles, 66°C down to 58°C (max 0.2°C/s) 15-60 seconds, 58°C 50 seconds, 65°C 6 minutes, 65°C 10 minute 1 cycle, 4°C hold.

Beads purification (0.8X)

1. Add 40 pl beads to each of well.

2. Mix 50 pl sample with 40 pl beads by pipetting 10 times, bind for 15 minutes. 3. Separate beads at magnetic rack for 5 min.

4. Remove buffer by slow pipetting.

5. Add lOOOpl 80% EtOH to each tube. After about 30 sec, slowly remove the EtOH by pipetting.

6. Repeat previous step two more times for a total of three times.

7. Let air dry, with the lids open, until you see the first signs of cracking (10-15 minutes).

8. Suspend beads in 23 pl water (RNase/DNase free).

9. Incubate for 2 minutes (not on magnetic rack).

10. Place tubes on magnetic rack for 5 minutes.

11. Collect 21 pl from each tube on the magnetic rack.

12. Store material in -20°C.

End Tailing (NEB Ultra II End Prep Enzyme Mix)

1. Flick and/or invert the reagent tubes to ensure they are well mixed.

2. Always spin down tubes before opening for the first time each day.

3. Transfer 200 frnol of cDNA amplicons into a clean 0.2 ml thin-walled PCR tube and adjust the volume to 50 pl with nuclease-free water.

4. In the same 0.2 ml thin-walled PCR tube, mix the following. Between each addition, pipette mix 10-20 times: cDNA amplicons 50 pl, Ultra II End-prep Reaction Buffer 7 pl, Ultra II End-prep Enzyme Mix 3 pl (total 60 pl).

5. Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes. 6. Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.

Beads purification (0.6X)

1. Add 40 pl beads to each of well.

2. Mix 60 pl sample with 40 pl beads by pipetting 10 times, bind for 15 min.

3. Separate beads at magnetic rack for 5 min.

4. Remove buffer by slow pipetting; make sure not to touch the beads.

5. Add lOOOpl 80% EtOH to each tube. After about 30 sec, slowly remove the EtOH by pipetting.

6. Repeat previous step two more times for a total of three times.

7. Let air dry, with the lids open, until you see the first signs of cracking (10-15 min). Do not over dry the samples!

8. Suspend beads in 63 pl water (RNase/DNase free). In this step you have to touch the beads with the pipette tip.

9. Incubate for 2 min (not on magnetic rack).

10. Place tubes on magnetic rack for 5 min.

11. Collect 60 pl from each tube on the magnetic rack.

12. Store material in -20C. Adaptor ligation

1. Spin down the Ligation Adapter (LA) and Quick T4 Ligase, and place on ice.

2. Thaw Ligation Buffer (LNB) at room temperature, spin down and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.

3. Thaw the Elution Buffer (EB) at room temperature and mix by vortexing. Then spin down and place on ice.

4. Thaw the Short Fragment Buffer (SFB) at room temperature and mix by vortexing. Then spin down and place on ice.

5. In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order: DNA sample from the previous step 60 pl, Ligation Buffer (LNB) 25 pl, NEBNext Quick T4 DNA Ligase 10 pl, Ligation Adapter (LA) 5 pl.

6. Thoroughly mix the reaction by gently pipetting and briefly spinning down.

Incubate the reaction for 10 minutes at room temperature.

Bead purification (0.4X)

1. Add 40 pl beads to each of well.

2. Mix 100 pl sample with 40 pl beads by pipetting 10 times, bind for 15 minutes. 3. Separate beads at magnetic rack for 5 minutes.

4. Remove buffer by slow pipetting.

5. Add 1000 pl 80% EtOH to each tube. After about 30 seconds, slowly remove the EtOH by pipetting.

6. Repeat previous step two more times for a total of three times.

7. Let air dry, with the lids open, until you see the first signs of cracking (10-15 minutes).

8. Suspend beads in 34 pl water (RNase/DNase free).

9. Incubate for 2 minutes (not on magnetic rack).

10. Place tubes on magnetic rack for 5 minutes.

11. Collect 32 pl from each tube on the magnetic rack.

12. Store material in -20°C. All patent and non-patent publications cited in this specification are incorporated herein by reference in their entireties. US Provisional Patent Application No. 63/710,352, filed October 22, 2025, is incorporated herein by reference. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

What is claimed is:

1. A solid substrate for performing spatially resolved transcriptome sequencing of one or more biological specimens, the solid substrate having a surface comprising:

an array of capture regions comprising substrate oligonucleotides bound to the solid substrate, the substrate oligonucleotides comprising a cleavable motif, a UMI, a first sector barcode, a second sector barcode, a first pixel barcode, a second pixel barcode, and a capture sequence, each capture region of the array being identifiable by the substrate oligonucleotide bound thereto;

wherein the first sector barcode is common to the substrate oligonucleotides of a series of adjacent rows of capture regions of the array, and the second sector barcode is a common to the substrate oligonucleotides of a series of adjacent columns of capture regions of the array, each combination of first sector barcode and second sector barcode defining a sector; and

each sector comprising multiple capture regions, each capture region comprising substrate oligonucleotides comprising a unique combination of first pixel barcode and second pixel barcode, wherein each unique combination of first pixel barcode and second pixel barcode defines x and y positions, respectively, within that sector.

2. A solid substrate for performing spatially resolved transcriptome sequencing of one or more biological specimens, the solid substrate having a surface comprising:

an array of capture regions comprising substrate oligonucleotides bound to the solid substrate, the substrate oligonucleotides comprising a cleavable motif, a UMI, a first pixel barcode, a second pixel barcode, a first sector barcode, a second sector barcode, and a capture sequence, each capture region of the array being identifiable by the substrate oligonucleotide bound thereto;

wherein the first sector barcode is common to the substrate oligonucleotides of a series of adjacent rows of capture regions of the array, and the second sector barcode is a common to the substrate oligonucleotides of a series of adjacent columns of capture regions of the array, each combination of first sector barcode and second sector barcode defining a sector; and

each sector comprising multiple capture regions, each capture region comprising substrate oligonucleotides comprising a unique combination of first pixel barcode and second pixel barcode, wherein each unique combination of first pixel barcode and second pixel barcode defines x and y positions, respectively, within that sector. 3. The solid substrate of claim 1 or 2, wherein the array of capture regions comprises substrate oligonucleotides having a plurality of unique first sector barcodes and a plurality of unique second sector barcodes.

4. The solid substrate of claim 3, wherein the array of capture regions comprises substrate oligonucleotides having at least four unique first sector barcodes and at least four unique second sector barcodes defining 16 sectors.

5. The solid substrate of claim 1 or 2 wherein the array of capture regions comprises substrate oligonucleotides having a plurality of unique first pixel barcodes and a plurality of unique second pixel barcodes.

6. The solid substrate of claim 5, wherein the array of capture regions comprises substrate oligonucleotides having about 96 unique first pixel barcodes spaced about 3 pm apart and about 96 unique second pixel barcodes spaced about 3 pm apart.

7. The solid substrate of claim 1 or 2, wherein the capture regions are spaced about 3 pm apart to about 200pm apart in any direction.

8. The solid substrate of claim 1 or 2, wherein the substrate oligonucleotides comprise linkers joining each of the first sector barcode, the second sector barcode, the first pixel barcode, and the second pixel barcode.

9. The solid substrate of claim 1 or 2, wherein the substrate oligonucleotides further comprise a linker joining the second pixel barcode and the capture sequence.

10. The solid substrate of claim 1 or 2, wherein the substrate oligonucleotides further comprise a primer handle.

11. The solid substrate of claim 1 or 2, further comprising an offset UMI anchor.

12. The solid substrate of claim 1 or 2, wherein the oligonucleotides further comprise a cleavable motif, optionally wherein the cleavable motif comprises i) a sequence of at least 3, 4, 5, or 6 deoxyuridine (dU) bases or ii) a restriction recognition site.

13. The solid substrate of claim 1 or 2, wherein the capture sequence is a poly(d)T or poly(d)T VN. 14. The solid substrate of claim 1 or 2, further comprising fiducial pixels comprising fiducial oligonucleotides bound to the solid substrate.

15. The solid substrate of claim 1 or 2, wherein the fiducial pixels further comprise a fluorescent label.

16. The solid substrate of claim 1 or 2, comprising 140,000 or more capture regions, optionally wherein the array of capture regions is an area of at least about 1 cm².

17. The solid substrate of claim 1 or 2, comprising an array of capture regions comprising up to about 9,200 unique capture regions in an area of about 9.2 mm² and/or up to about 1,800,000 unique capture regions in an area of about 8.0 mm².

18. The solid substrate of claim 1 or 2, wherein the solid substrate is a functionalized glass slide, optionally a glass slide that is 75 x 25 x 1 mm.

19. The solid substrate of claim 1 or 2, wherein the substrate oligonucleotides further comprise a UMI offset anchor, optionally wherein the UMI offset anchor is immediately 3’ to the UMI.

20. A method for generating a solid substrate having a surface comprising an array of capture regions, the capture regions being arranged in rows and columns, the method comprising:

i) contacting each row in a series of adjacent rows of a solid substrate with one of a set of first sector barcode oligonucleotides, each first sector barcode oligonucleotide comprising a first sector barcode and a first linker, wherein each first sector barcode oligonucleotide in the set has a different first sector barcode, wherein the first sector barcode oligonucleotides are bound to the solid substrate using a water-soluble amine linker, optionally bis(sulfosuccinimidyl)suberate (BS3), wherein the first sector barcode oligonucleotides comprise a 5’ amine modification;

ii) contacting each column in a series of adjacent columns of the solid substrate with one of a set of second sector barcode oligonucleotides, each second sector barcode oligonucleotide comprising a second sector barcode and a second linker, wherein each second sector barcode oligonucleotide in the set has a different second sector barcode, wherein each combination of first sector barcode and second sector barcode defines a sector, iii) contacting each row in the series of adjacent rows of the solid substrate with a set of first pixel barcode oligonucleotides, each first pixel barcode oligonucleotide comprising a first pixel barcode and a third linker, wherein each first pixel barcode oligonucleotide in the set has a different first pixel barcode;

iv) contacting each column in the series of adjacent columns of the solid substrate with a set of second pixel barcode oligonucleotides, each second pixel barcode oligonucleotide comprising a second pixel barcode and a fourth linker, wherein each second pixel barcode oligonucleotide in the set has a different second pixel barcode, wherein each combination of first pixel barcode and second pixel barcode defines x and y positions, respectively, within a sector; and

v) contacting each column and row of the solid substrate with a capture oligonucleotide comprising a fourth linker and a capture sequence.

21. A method for generating a solid substrate having a surface comprising an array of capture regions, the capture regions being arranged in rows and columns, the method comprising:

i) contacting each row in a series of adjacent rows of a solid substrate with one of a set of first sector barcode oligonucleotides, each first sector barcode oligonucleotide comprising a first sector barcode and a first linker, wherein each first sector barcode oligonucleotide in the set has a different first sector barcode, wherein the first sector barcode oligonucleotides are bound to the solid substrate having an amine-reactive functional groups, optionally N-oxysuccinimide, wherein the first sector barcode oligonucleotides comprise a 5’ amine modification;

ii) contacting each column in a series of adjacent columns of the solid substrate with one of a set of second sector barcode oligonucleotides, each second sector barcode oligonucleotide comprising a second sector barcode and a second linker, wherein each second sector barcode oligonucleotide in the set has a different second sector barcode, wherein each combination of first sector barcode and second sector barcode defines a sector,

iii) contacting each row in the series of adjacent rows of the solid substrate with a set of first pixel barcode oligonucleotides, each first pixel barcode oligonucleotide comprising a first pixel barcode and a third linker, wherein each first pixel barcode oligonucleotide in the set has a different first pixel barcode;

iv) contacting each column in the series of adjacent columns of the solid substrate with a set of second pixel barcode oligonucleotides, each second pixel barcode oligonucleotide comprising a second pixel barcode and a fourth linker, wherein each second pixel barcode oligonucleotide in the set has a different second pixel barcode, wherein each combination of first pixel barcode and second pixel barcode defines x and y positions, respectively, within a sector; and

v) contacting each column and row of the solid substrate with a capture oligonucleotide comprising a fourth linker and a capture sequence.

22. The method of claim 20 or 21, wherein the first sector barcode oligonucleotides comprise a cleavable motif, a UMI, and a first sector barcode, optionally further comprising a primer handle.

23. The method of any claim 22, wherein the cleavable motif cleavable motif comprises i) a sequence of at least 3, 4, 5, or 6 deoxyuridine (dU) bases or ii) a restriction recognition site.

24. The method of claim 20 or 21, wherein, after step i), the first sector barcode oligonucleotide is bound to the substrate.

25. The method of claim 24, wherein, after step i), the solid substrate is contacted with a set of first splint oligonucleotides, each first splint oligonucleotide in the set comprising a sequence complementary to the first linker and one of the second sector barcodes, whereby the first splint oligonucleotide is bound to the first sector oligonucleotide.

26. The method of claim 25, wherein after step ii), the second sector barcode oligonucleotide is bound to the first splint oligonucleotide.

27. The method of claim 21 to 22, wherein the first sector barcode oligonucleotides comprise a duplex region, the duplex region having an overhang having a sequence complementary to one of the second sector barcodes.

28. The method of claim 27, wherein after step ii), the solid substrate is contacted with a set of second splint oligonucleotides, each second splint oligonucleotide in the set comprising a sequence complementary to the second linker and one of the first pixel barcodes whereby the second splint oligonucleotide is bound to the second sector oligonucleotide.

29. The method of claim 28, wherein after step iii), the first pixel barcode oligonucleotide is bound to the second splint oligonucleotide. 30. The method of any one of claims 21 to 22, wherein the second sector barcode oligonucleotides comprise a duplex region, the duplex region having an overhang having a sequence complementary to one of the first pixel barcodes.

31. The method of claim 30, wherein after step iv), the solid substrate is contacted with a set of third splint oligonucleotides, each third splint oligonucleotide in the set comprising a sequence complementary to the third linker and one of the second pixel barcodes whereby the third splint oligonucleotide is bound to the first pixel barcode oligonucleotide.

32. The method of claim 31, wherein after step iv), the second pixel barcode oligonucleotide is bound to the third splint oligonucleotide.

33. The method of claim 21 or 22, wherein the third sector barcode oligonucleotides comprise a duplex region, the duplex region having an overhang having a sequence complementary to one of the second pixel barcodes.

34. The method of claim 33, wherein after step v), the solid substrate is contacted with a fourth splint oligonucleotide comprising a sequence complementary to the fourth linker and the capture oligonucleotide whereby the fourth splint oligonucleotide is bound to the second pixel barcode oligonucleotide.

35. The method of claim 34, wherein after step v), the capture oligonucleotide is bound to the fourth splint oligonucleotide.

36. The method of claim 21 or 22, wherein the fourth sector barcode oligonucleotides comprise a duplex region, the duplex region having an overhang having a sequence complementary to the capture sequence.

37. The method of any one of claims 20 to 36, wherein the capture sequence is a poly(d)T or poly(d)T VN.

38. The method of any one of claims 20 to 37, further comprising, after step v), performing a ligation reaction.

39. The method of any one of claims 20 to 38, further comprising performing a ligation reaction after step i), ii), iii), iv), and/or v). 40. The method of any one of claims 20 to 39, wherein ligation comprises addition of T4 ligase.

41. The method of any one of claims 20 to 40, further comprising removing the splint oligonucleotides.

42. The method of any one of claims 20 to 41, wherein in step i)

a) first sector barcode oligonucleotides are present at a concentration of about 0.1 pM to about 10 pM; and/or

b) the water-soluble amine linker is present at a concentration of about 2.5 mM.

43. A solid substrate obtained by the method of any one of claims 20 to 42.

44. A method of binding oligonucleotides to a solid substrate, the method comprising binding the oligonucleotides to the solid substrate using a water-soluble amine linker, optionally bis(sulfosuccinimidyl)suberate, wherein the oligonucleotides comprise a 5’ amine modification.

45. The method of claim 44, wherein

a) the oligonucleotides are present at a concentration of about 1 to about lOpM; and/or

b) the water-soluble amine linker is present at a concentration of about 2.5 mM.

46. A solid substrate obtained by the method of claim 44 or 45.

47. A method of spatially resolved transcriptome sequencing of one or more biological specimens, the method comprising:

a) providing the solid substrate of any one of claims 1 to 19 or 43;

b) mounting the one or more biological samples to the solid substrate, the biological samples partially or completely overlaying the array of capture regions;

c) performing staining or immunofluorescent labeling of the one or more biological samples;

d) capturing one or more images of the one or more biological samples;

e) permeabilizing the one or more biological specimens thereby permitting RNA present in the one or more biological specimens to bind capture sequences of the substrate oligonucleotides; f) performing reverse-transcription by addition of a reverse transcriptase under conditions suitable to permit generation of cDNA:RNA hybrid molecules, wherein the reverse transcription includes extension of RNA using the bound substrate oligonucleotide as a template to result in tagging of the RNA with sequences complementary to the first sector barcode, the second sector barcode, the first pixel barcode, and the second pixel barcode of the bound substrate oligonucleotide; and

g) sequencing tagged RNA and assigning sequences to a capture region.

48. The method of claim 47, wherein the solid substrate is contacted with fluorescently tagged oligonucleotide that hybridize with the fiducial pixels.

49. The method of claim 47 or 48, wherein the reverse transcriptase is a hybrid reverse transcriptase-helicase enzyme.

50. The method of any one of claims 47 to 49, wherein the reverse transcriptase is a modified MMLV reverse transcriptase (RT), Maxima H Minus Reverse Transcriptase, SuperScript™ II Reverse Transcriptase, Induro® Reverse Transcriptase, M-MLV Reverse Transcriptase, UltraMarathonRT (RNA Connect).

51. The method of any one of claims 47 to 50, further comprising releasing the substrate oligonucleotides bound to the solid substrate by addition of an enzyme that cleaves the cleavable motif of the oligonucleotides.

52. The method of any one of claims 49 to 51, wherein the enzyme that cleaves the cleavable motif is added following step f).

53. The method of any one of claims 49 to 52, wherein step f) further comprises addition of an enzyme that cleaves the cleavable motif of the oligonucleotides.

54. The method of any one of claims 49 to 53, further comprising adapter ligation, wherein a ligase enzyme covalently attaches a sequencing adapter to the RNA of the cDNA:RNA hybrid molecules, and performing sequencing of the RNA, optionally wherein the sequencing is long run sequencing and/or nanopore sequencing.

55. The method of any one of claims 49 to 54, wherein the capture sequences are poly(d)T or poly(d)T VN. 56. The method of any one of claims 44 to 54, wherein up to 16 biological samples are mounted to the array, each sample being mounted to a sector of the array identifiable by a combination of the first sector barcode and the second sector barcode.

57. The method of any one of claims 49 to 56, wherein the method does not comprise second strand cDNA synthesis or PCR amplification.