Next-generation volumetric in situ sequencing

Abstract

Provided herein are devices, methods, and systems for next-generation volumetric in situ sequencing of nucleic acids in cells in intact tissues. In particular, methods are provided for improving robustness across sample types, including thin and thick tissue volumes, and increasing the efficiency and specificity of target labeling for volumetric combined in situ sequencing.

Description

[Background technology]

[0001] Biological samples contain complex and heterogeneous genetic information that spans the length scale of individual cells to the length scale of entire tissues. Spatial patterns of nucleic acids within cells may reveal properties and abnormalities of cell function, cumulative distribution of RNA expression may define cell type or function, and systematic variations in the location of cell types within tissues may define tissue function. The combination of anatomical connectivity information encoded in nucleic acids and cell type distribution throughout tissues may span many sections of tissue. Thus, techniques for in situ nucleic acid sequencing must be able to bridge resolutions as small as individual molecules and as large as the entire brain. To efficiently collect and record this information across orders of magnitude difference in length, new inventions are needed to enhance the robustness, rapidity, automation, and high-throughput nature of in situ sequencing techniques. Summary of the Invention

[0002] Provided herein are devices, methods, and systems for next-generation volumetric in situ sequencing of nucleic acids in cells in intact tissues. In particular, methods are provided for improving robustness across sample types, including thin and thick tissue volumes, and increasing the efficiency and specificity of target labeling for volumetric sequential and combinatorial in situ sequencing.

[0003] In one aspect, a method is provided for in situ genetic sequencing of a target nucleic acid in cells in an intact tissue, the method comprising: (a) contacting a fixed, permeabilized intact tissue with at least a pair of oligonucleotide primers under conditions permitting specific hybridization, the pair of primers comprising a first oligonucleotide and a second oligonucleotide, each of the first oligonucleotide and the second oligonucleotide comprising a first region of complementarity, a second region of complementarity, and a third region of complementarity, and the second oligonucleotide comprising a first region of complementarity, and a second region of complementarity, the second oligonucleotide comprising a second region of complementarity, and the first region of complementarity, the second region of complementarity, and the second ... the second region of complementarity, and the second region of complementarity, the second region of complementarity, the second region of complementarity, and the third region of complementarity. wherein the first complementary region of the first oligonucleotide is complementary to a first portion of the target nucleic acid, the second complementary region of the first oligonucleotide is complementary to the first complementary region of the second oligonucleotide, the third complementary region of the first oligonucleotide is complementary to the third complementary region of the second oligonucleotide, the second complementary region of the second oligonucleotide is complementary to a second portion of the target nucleic acid, the first portion of the target nucleic acid is adjacent to the second portion of the target nucleic acid, and the second complementary region of the first oligonucleotide is complementary to a barcode sequence. (b) adding a ligase to ligate the second oligonucleotide to generate a closed nucleic acid circle; (c) performing rolling circle amplification in the presence of the nucleic acid molecule, the rolling circle amplification comprising forming one or more amplicons using the second oligonucleotide as a template and the first oligonucleotide as a primer for a polymerase; (d) embedding the one or more amplicons in the presence of a hydrogel subunit to form one or more hydrogel-embedded amplicons; and (e) contacting the one or more hydrogel-embedded amplicons having the barcode sequence with a set of sequencing primers under conditions that allow for ligation,(f) contacting the set of sequencing primers, the set comprising a third oligonucleotide configured to decode bases and a fourth oligonucleotide configured to convert the decoded bases into a signal, and ligation occurs only when both the third oligonucleotide and the fourth oligonucleotide are complementary to adjacent sequences of the same amplicon; (g) imaging the amplicons embedded in one or more hydrogels to in situ determine the genetic sequence of the target nucleic acid in cells in the intact tissue;

[0004] In certain embodiments, the method further comprises contacting the fixed, permeabilized intact tissue with a gel adaptor oligonucleotide that binds to a first oligonucleotide, the gel adaptor oligonucleotide comprising a nucleotide modification at the 5' or 3' end that links the gel adaptor oligonucleotide to a hydrogel during gelation. In some embodiments, the nucleotide modification comprises an acrydite group. In some embodiments, the first oligonucleotide further comprises a common binding site for the gel adaptor oligonucleotide. In some embodiments, the common binding site for the gel adaptor oligonucleotide is adjacent to the first complementary region of the first oligonucleotide.

[0005] In certain embodiments, the method further comprises contacting the fixed, permeabilized intact tissue with an oligonucleotide probe for amplicon detection and condensation, where the oligonucleotide probe binds to a second oligonucleotide. In some embodiments, the second oligonucleotide further comprises a common binding site for the oligonucleotide probe for amplicon detection and condensation. In some embodiments, the common binding site for the oligonucleotide probe for amplicon detection and condensation is adjacent to a second complementary region or adjacent to a sequence that is complementary to the second half of the unique matching sequence of the second oligonucleotide. In some embodiments, the oligonucleotide probe comprises a unique sequence for detecting the amplicon of the intended probe target and two or more copies of a sequence that is complementary to a common sequence on the amplicon. In some embodiments, the oligonucleotide probe further comprises a nucleotide modification at the 5' or 3' end such that the first oligonucleotide probe is linked to the hydrogel during gelation. In some embodiments, the nucleotide modification comprises an acrydite group.

[0006] In certain embodiments, the method further comprises barcoding the cells by contacting the cells with i) a first probe comprising a 5' amine modification or a 5' biotin modification, a common gel adaptor complementary sequence that hybridizes to a gel adaptor oligonucleotide, and a unique barcode sequence, and ii) a second probe comprising a first sequence that is complementary to a first portion of the unique barcode sequence and a second sequence that is complementary to a second portion of the unique barcode sequence, wherein the first sequence and the second sequence are adjacent to a sequencing encoding sequence, and hybridization of the first probe and the second probe results in the formation of a barcode complex comprising the first probe and the second probe. In some embodiments, the second probe is a padlock probe.

[0007] In certain embodiments, the first portion of the target nucleic acid and the second portion of the target nucleic acid have approximately the same melting temperature.

[0008] In certain embodiments, the method further comprises contacting the fixed, permeabilized intact tissue with an mRNA-holding oligonucleotide, the mRNA-holding oligonucleotide comprising a nucleotide modification at the 5' or 3' end, where the mRNA-holding oligonucleotide is linked to the hydrogel during gelation, and a poly-T tail that hybridizes to the poly-A tail of the mRNA, where hybridization of the poly-T tail of the mRNA-holding oligonucleotide with the poly-A tail of the mRNA retains the mRNA in the hydrogel, and a unique hybridization sequence. In some embodiments, the poly-T tail comprises interleaved locked nucleic acid (LNA) thymine (T) bases. In some embodiments, the method further comprises contacting the fixed, permeabilized intact tissue with a fluorescently labeled probe oligonucleotide that selectively binds to the unique hybridization sequence of the mRNA-holding oligonucleotide.

[0009] In certain embodiments, the unique matching sequence is a randomized sequence.

[0010] In certain embodiments, sequencing is performed using sequential or combinatorial encoding.

[0011] In certain embodiments, the method further comprises pre-incubating the tissue sample with a polymerase for a period of time sufficient to allow uniform diffusion of the polymerase throughout the tissue prior to performing rolling circle amplification.

[0012] In certain embodiments, the signal is a fluorescent signal.

[0013] In certain embodiments, imaging is performed in the presence of an anti-fading buffer that includes an antioxidant, for example, the anti-fading buffer can include, but is not limited to, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and Trolox-quinone.

[0014] In certain embodiments, the method further comprises removing the signal after imaging by contacting the hydrogel with formamide.

[0015] In certain embodiments, the fourth oligonucleotide is covalently linked to the fluorophore by a disulfide bond. In some embodiments, the method further comprises contacting the hydrogel after said imaging with a reducing agent, where reduction of the disulfide bond results in cleavage of the fluorophore from the fourth oligonucleotide.

[0016] In certain embodiments, ligation of the third oligonucleotide and the fourth oligonucleotide is carried out in the presence of a polyethylene glycol polymer (e.g., PEG6000).

[0017] In certain embodiments, the oligoconjugate to the target binding agent is detected by hybridization of an oligonucleotide containing a fluorophore, hi certain embodiments, this signal is removed after detection by competitive binding of a displacing oligonucleotide.

[0018] In another aspect, a method is provided for screening a candidate agent to determine whether it modulates gene expression of a nucleic acid in a cell in an intact tissue, the method comprising performing a method described herein to determine the gene sequence of a target nucleic acid in a cell in the intact tissue, and detecting a level of gene expression of the target nucleic acid, wherein a change in the level of expression of the target nucleic acid in the presence of the candidate agent relative to the level of expression of the target nucleic acid in the absence of the candidate agent indicates that the candidate agent modulates gene expression of a nucleic acid in a cell in the intact tissue.

[0019] In certain embodiments, the detecting comprises performing flow cytometry, sequencing, probe binding and electrochemical detection, pH changes, catalysis induced by enzymes bound to DNA tags, quantum entanglement, Raman spectroscopy, terahertz wave technology, and / or scanning electron microscopy. In some embodiments, the flow cytometry is mass cytometry or fluorescence activated flow cytometry. In some embodiments, the detecting comprises performing microscopy, scanning mass spectrometry, or other imaging techniques. In some embodiments, the detecting comprises detecting a signal, such as a fluorescent signal.

[0020] In another aspect, a system is provided comprising a fluidic device and a processor unit configured to perform the methods described herein for in situ genetic sequencing of target nucleic acids in cells in intact tissue.

[0021] In certain embodiments, the system further comprises an imaging chamber.

[0022] In certain embodiments, the system further comprises a pump. [Brief description of the drawings]

[0023] [Figure 1A]Next-generation STARmap2 signal across a thick-section (200um) mouse brain tissue sample. Maintaining the section floating throughout the sample preparation, combined with dextran in the hybridization solution, PEG in the ligation solution, and pre-incubation with inactive RCA polymerase solution, results in consistent, highly efficient and uniform labeling across the entire sample Z-axis. [Figure 1B] Next-generation STARmap2 signal across a thick-section (200um) mouse brain tissue sample. Maintaining the section floating throughout the sample preparation, combined with dextran in the hybridization solution, PEG in the ligation solution, and pre-incubation with inactive RCA polymerase solution, results in consistent, highly efficient and uniform labeling across the entire sample Z-axis. [Figure 1C] Next-generation STARmap2 signal across a thick-section (200um) mouse brain tissue sample. Maintaining the section floating throughout the sample preparation, combined with dextran in the hybridization solution, PEG in the ligation solution, and pre-incubation with inactive RCA polymerase solution, results in consistent, highly efficient and uniform labeling across the entire sample Z-axis. [Figure 1D] Next-generation STARmap2 signal across a thick-section (200um) mouse brain tissue sample. Maintaining the section floating throughout the sample preparation, combined with dextran in the hybridization solution, PEG in the ligation solution, and pre-incubation with inactive RCA polymerase solution, results in consistent, highly efficient and uniform labeling across the entire sample Z-axis. [Diagram 2] Figure 1 shows efficient and high signal-to-noise ratio (SNR) labeling with STARmap2 in a postmortem human brain tissue sample. A single readout channel (488) of four acquisition channels is shown in a single optical plane from the first combined round of sequencing. The dashed boxes highlight individual cells within the tissue. The dot signals are amplicons. [Diagram 3]1 shows the workflow for STARmap2, where samples are kept floating through hybridization, clarification, ligation, and RCA reactions before being recombined in preparation for sequencing. [Figure 4] Combinatorial chemical sequencing of 200 genes in a 150um thick mouse brain section. Data are presented showing a single optical plane from a single combinatorial round of labeling. Left: a single XY optical plane, top right: a ZY optical plane showing consistent labeling efficiency across the Z axis with an overall high SNR, bottom right: detail on multiple cells in a single XY plane. [Diagram 5] The effect of incorporating a unique sequence at the probe circularization junction is shown. (A, B) Samples were prepared with a common set of STARmap2 probes against Actb and a 384 probe targeting a random (exogenous) sequence not contained within the sample. (A) Samples labeled with 384 probes targeting random sequences contained unique (probe-spanning) sequences at their circularization junctions. Unique sequences were generated by randomizing sequences and filtering by cross-sequence similarity and melting temperature. (B) Sample labeled with 384 probes targeting the same set of random (exogenous) sequences as in (A), except that the probes contain constant sequences at the circularization junctions, similar to the original SNAIL probe design. Insets at the bottom right of (A) and (B) showing details in the intercellular space. Dashed boxes indicate inset location. Samples prepared in parallel from adjacent tissue sections. Images taken at 20x magnification from a single brightest plane with equal imaging and contrast settings are shown. [Figure 6] An overview of STARmap2 probe modifications (excluding sequence details used for improved sequencing chemistry) is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Provided herein are devices, methods, and systems for next-generation volumetric in situ sequencing of nucleic acids in cells in intact tissues. In particular, methods are provided for improving robustness across sample types, including thin and thick tissue volumes, and increasing the efficiency and specificity of target labeling for volumetric combined in situ sequencing.

[0025] Before the present devices, methods, and systems are described, it is to be understood that this invention is not limited to the particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, since the scope of the present invention will be limited only by the appended claims.

[0026] Where a range of values ​​is provided, unless the context dictates otherwise, it is understood that each intervening value between the upper and lower limits of that range is also specifically disclosed to the tenth of the unit of the lower limit. Each smaller range between any stated value or intervening value within a stated range and any other stated value or intervening value within that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range in which either, neither, or both limits are included in the smaller range is also encompassed within the invention, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are likewise included in the invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, it should be understood that the present disclosure supersedes any disclosure of the incorporated publication.

[0028] As will be apparent to those skilled in the art upon reading this disclosure, each of the separate embodiments described and illustrated herein has separate components and features which may be readily separated from or combined with the features of any of the other various embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0029] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the peptide" includes a reference to one or more peptides and equivalents thereof known to those skilled in the art, such as oligopeptides or polypeptides.

[0030] Publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0031] definition The term "about" is meant to encompass a deviation of plus or minus 5 percent, particularly in relation to a given amount.

[0032] The terms "peptide," "oligopeptide," "polypeptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. Both full-length proteins and fragments thereof are encompassed by the definition. The term also includes post-expression modifications of the polypeptide, such as phosphorylation, glycosylation, acetylation, hydroxylation, oxidation, etc., as well as chemically or biochemically modified or derivatized amino acids and polypeptides having modified peptide backbones. The term also includes fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences with or without an N-terminal methionine residue, immunologically tagged proteins, and the like. The term includes polypeptides that include one or more of a fatty acid moiety, a lipid moiety, a sugar moiety, and a carbohydrate moiety.

[0033] As used herein, the term "target nucleic acid" refers to any polynucleotide nucleic acid molecule (e.g., DNA molecule, RNA molecule, modified nucleic acid, etc.) present within a single cell. In some embodiments, the target nucleic acid is a coding RNA (e.g., mRNA). In some embodiments, the target nucleic acid is a non-coding RNA (e.g., tRNA, rRNA, microRNA (miRNA), mature miRNA, immature miRNA, etc.). In some embodiments, the target nucleic acid is a splice variant of an RNA molecule (e.g., mRNA, pre-mRNA, etc.) in the context of a cell. Thus, suitable target nucleic acids can be unspliced ​​RNA (e.g., pre-mRNA, mRNA), partially spliced ​​RNA, or fully spliced ​​RNA, etc. Target nucleic acids of interest may be variably expressed, i.e., have different abundances, within a population of cells, and the methods of the present invention allow for profiling and comparison of expression levels of nucleic acids, including but not limited to RNA transcripts, in individual cells. Target nucleic acids may also be DNA molecules, such as modified genomes, viruses, plasmids, etc. For example, the methods may be used to detect copy number variants, e.g., in cancer cell populations where a target nucleic acid is present at different abundances in the genomes of cells in the population, or in virally infected cells to determine viral load and kinetics.

[0034] The terms "oligonucleotide", "polynucleotide" and "nucleic acid molecule", used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide may contain sugar and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide may contain polymers of synthetic subunits, such as phosphoramidites and / or phosphorothioates, and thus may be oligodeoxynucleoside phosphoramidates or mixed phosphoramidate-phosphodiester oligomers. See Peyrottes et al. (1996) Nucl. Acids Res. 24:1841-1848, Chaturvedi et al. (1996) Nucl. Acids Res. 24:2318-2323. A polynucleotide may contain one or more L nucleosides. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars, and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be modified to contain N3'-P5' (NP) phosphoramidates, morpholinophosphocyamidates (MF), locked nucleic acids (LNA), 2'-O-methoxyethyl (MOE), or 2'-fluoroarabinonucleic acid (FANA), which can enhance the resistance of the polynucleotide to nuclease degradation (see, e.g., Faria et al. (2001) Nature Biotechnol. 19:40-44; Toulme (2001) Nature Biotechnol. 19:17-18).Polynucleotides may be further modified after polymerization, for example, by conjugation with a labeling moiety. Other types of modifications included in this definition are capping, substitution with one or more analogs of naturally occurring nucleotides, and the introduction of a means for binding the polynucleotide to a protein, a metal ion, a labeling moiety, another polynucleotide, or a solid support. The immune modulatory nucleic acid molecule may be provided in various formulations, for example, in association with liposomes, microencapsulation, etc., as described in more detail herein. The polynucleotides used for amplification are generally single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the polynucleotide is first treated to separate its strands, which can then be used to prepare extension products. This denaturation step is typically affected by heat, but may alternatively be carried out using alkali, followed by neutralization.

[0035] "Isolated," when referring to a protein, polypeptide, or peptide, means that the indicated molecule is separated and separate from the whole organism in which it is found in nature, or exists in the substantial absence of other biological macromolecules of the same type. With reference to polynucleotides, the term "isolated" refers to a nucleic acid molecule that lacks all or part of a sequence that is normally associated with it in nature, or a sequence that is naturally occurring but has heterologous sequences associated with it, or a molecule that is dissociated from the chromosome.

[0036] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to invertebrate and vertebrate animals, including, but not limited to, arthropods (e.g., insects, crustaceans, arachnids), cephalopods (e.g., octopus, squid), amphibians (e.g., frogs, salamanders, caecilians), fish, reptiles (e.g., turtles, crocodiles, snakes, earthworms, lizards, tuatara), mammals, including humans and non-human mammals, such as chimpanzees and other apes and non-human primates, including monkey species, laboratory animals, such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas, domestic animals, such as dogs and cats, farm animals, such as sheep, goats, pigs, horses, and cows, and birds, such as chickens, turkeys, and other pheasants, ducks, and geese, domestic, wild, and game birds. In some cases, the methods of the invention find use in laboratory animals, in veterinary applications, and in the development of animal models for disease, including, but not limited to, rodents, including mice, rats, and hamsters, primates, and transgenic animals.

[0037] method The methods disclosed herein include the use of a modified spatially resolved transcription amplicon read mapping (STARmap) technique, referred to as "STARmap2". For a description of the original STARmap technique, see, for example, International Patent Application Publication No. 2019 / 199579A1 and Wang et al. (2018) Science 361(6400):eaat5691, which are incorporated herein by reference in their entireties. Similar to STARmap, STARmap2 utilizes image-based in situ nucleic acid (DNA and / or RNA) sequencing technology that uses a sequencing-by-ligation process, specific signal amplification, hydrogel histochemistry to convert biological tissues into transparent sequencing chips, and an associated data analysis pipeline to spatially resolve highly multiplexed gene detection at the subcellular and cellular levels. STARmap2 adds improvements in probe design to reduce background labeling, the use of adapter oligonucleotides for better labeling and retention of nucleic acid targets in the gel, reduces imaging cycling times, and offers the ability to perform either forward or reverse sequencing with highly efficient combinatorial barcode reading.

[0038] As summarized above, methods disclosed herein include methods for in situ genetic sequencing of a target nucleic acid in cells within an intact tissue, the method comprising: (a) contacting the fixed, permeabilized intact tissue with at least a pair of oligonucleotide primers under conditions permitting specific hybridization, the pair of primers comprising a first oligonucleotide and a second oligonucleotide, each of the first oligonucleotide and the second oligonucleotide comprising a first region of complementarity, a second region of complementarity, and a third region of complementarity, the second oligonucleotide further comprising a barcode sequence, the first region of complementarity of the first oligonucleotide being complementary to a first portion of the target nucleic acid, the second region of complementarity of the first oligonucleotide being complementary to the first region of complementarity of the second oligonucleotide, the third region of complementarity of the first oligonucleotide being complementary to the third region of complementarity of the second oligonucleotide, the second region of complementarity of the second oligonucleotide being complementary to a second portion of the target nucleic acid, and a second complementary region of the first oligonucleotide comprising a first portion of a unique matching sequence, a third complementary region of the first oligonucleotide comprising a second portion of a unique matching sequence, a first complementary region of the second oligonucleotide comprising a sequence that is complementary to the first portion of the unique matching sequence, and a third complementary region of the second oligonucleotide comprising a sequence that is complementary to the second portion of the unique matching sequence; (b) adding a ligase to ligate the second oligonucleotide to generate a closed nucleic acid circle; (c) performing rolling circle amplification in the presence of the nucleic acid molecule, comprising forming one or more amplicons using the second oligonucleotide as a template and the first oligonucleotide as a primer for a polymerase; (d) embedding the one or more amplicons in the presence of a hydrogel subunit to form one or more hydrogel-embedded amplicons; and (e) under conditions that permit ligation.(f) contacting the one or more hydrogel-embedded amplicons having barcode sequences with a set of sequencing primers, the set of sequencing primers including a third oligonucleotide configured to decode bases and a fourth oligonucleotide configured to convert the decoded bases into a signal, and ligation occurs only when both the third oligonucleotide and the fourth oligonucleotide are complementary to adjacent sequences of the same amplicon; (f) repeating step (e) multiple times; and (g) imaging the one or more hydrogel-embedded amplicons to determine in situ the genetic sequence of the target nucleic acid in cells in the intact tissue.

[0039] In certain embodiments, the method further comprises contacting the fixed, permeabilized intact tissue with a gel adaptor oligonucleotide that binds to a first oligonucleotide, the gel adaptor oligonucleotide comprising a nucleotide modification at the 5' or 3' end that links the gel adaptor oligonucleotide to a hydrogel during gelation. In some embodiments, the nucleotide modification comprises an acrydite group. In some embodiments, the first oligonucleotide further comprises a common binding site for the gel adaptor oligonucleotide. In some embodiments, the common binding site for the gel adaptor oligonucleotide is adjacent to the first complementary region of the first oligonucleotide.

[0040] In certain embodiments, the method further comprises contacting the fixed, permeabilized intact tissue with an oligonucleotide probe for amplicon detection and condensation, where the oligonucleotide probe binds to a second oligonucleotide. In some embodiments, the second oligonucleotide further comprises a common binding site for the oligonucleotide probe for amplicon detection and condensation. In some embodiments, the common binding site for the oligonucleotide probe for amplicon detection and condensation is adjacent to a second complementary region or adjacent to a sequence that is complementary to the second half of the unique matching sequence of the second oligonucleotide. In some embodiments, the oligonucleotide probe comprises a unique sequence for detecting the amplicon of the intended probe target and two or more copies of a sequence that is complementary to a common sequence on the amplicon. In some embodiments, the oligonucleotide probe further comprises a nucleotide modification at the 5' or 3' end such that the first oligonucleotide probe is linked to the hydrogel during gelation. In some embodiments, the nucleotide modification comprises an acrydite group.

[0041] In certain embodiments, the method further comprises barcoding the cells by contacting the cells with i) a first probe comprising a 5' amine modification or a 5' biotin modification, a common gel adaptor complementary sequence that hybridizes to a gel adaptor oligonucleotide, and a unique barcode sequence, and ii) a second probe comprising a first sequence that is complementary to a first portion of the unique barcode sequence and a second sequence that is complementary to a second portion of the unique barcode sequence, wherein the first sequence and the second sequence are adjacent to a sequencing encoding sequence, and hybridization of the first probe and the second probe results in the formation of a barcode complex comprising the first probe and the second probe. In some embodiments, the second probe is a padlock probe.

[0042] In certain embodiments, the method further comprises contacting the fixed, permeabilized intact tissue with an mRNA-holding oligonucleotide, the mRNA-holding oligonucleotide comprising a nucleotide modification at the 5' or 3' end, where the mRNA-holding oligonucleotide is linked to the hydrogel during gelation, and a poly-T tail that hybridizes to the poly-A tail of the mRNA, where hybridization of the poly-T tail of the mRNA-holding oligonucleotide with the poly-A tail of the mRNA retains the mRNA in the hydrogel, and a unique hybridization sequence. In some embodiments, the poly-T tail comprises a reciprocal locked nucleic acid (LNA) thymine (T) base. In certain embodiments, the method further comprises contacting the fixed, permeabilized intact tissue with a fluorescently labeled probe oligonucleotide that selectively binds to the unique hybridization sequence of the mRNA-holding oligonucleotide.

[0043] The methods disclosed herein also provide a method of screening a candidate agent to determine whether a candidate agent modulates gene expression of a nucleic acid in a cell in an intact tissue by performing the methods described herein to determine the gene sequence of a target nucleic acid in a cell in an intact tissue and detecting the level of gene expression of the target nucleic acid, where a change in the level of expression of the target nucleic acid in the presence of the candidate agent relative to the level of expression of the target nucleic acid in the absence of the candidate agent indicates that the candidate agent modulates gene expression of a nucleic acid in a cell in an intact tissue.

[0044] In certain aspects, the methods disclosed herein provide faster processing times, higher multiplexing (up to 1000 genes), higher efficiency, higher sensitivity, lower error rates, and more spatially resolved cell types compared to existing gene expression analysis tools. In such aspects, the improved hydrogel histochemistry transforms biological tissues into hydrogel-imprinted nucleic acids that are compatible with in situ sequencing, which is an improved sequencing by ligation technique (SCAL and SEDAL2) for in situ sequencing with reduced error. In some other aspects, the methods disclosed herein include spatial sequencing (e.g., reagents, chips, or services) for biomedical research and clinical diagnostics (e.g., cancer, bacterial infection, viral infection, etc.) with single cell and / or single molecule sensitivity.

[0045] Specific Amplification of Nucleic Acids via Intramolecular Ligation (SNAIL) In some embodiments, one component of STARmap2 comprises an efficient approach for generating cDNA libraries in situ from cellular RNA for specific amplification of nucleic acids via intramolecular ligation, which may be referred to as SNAIL. In certain embodiments, the methods of the invention comprise contacting fixed, permeabilized intact tissue with at least a pair of oligonucleotide primers under conditions that allow specific hybridization, the pair of primers comprising a first oligonucleotide and a second oligonucleotide.

[0046] More generally, the nucleic acid present in the cells of interest in the tissue serves as a scaffold for the assembly of a complex comprising a pair of primers, herein referred to as a first oligonucleotide and a second oligonucleotide. In some embodiments, contacting the fixed and permeabilized intact tissue comprises hybridizing a pair of primers to the same target nucleic acid. In some embodiments, the target nucleic acid is RNA. In such embodiments, the target nucleic acid may be mRNA. In other embodiments, the target nucleic acid is DNA.

[0047] As used herein, the terms "hybridize" and "hybridization" refer to the formation of a complex between nucleotide sequences that are sufficiently complementary to form a complex through Watson-Crick base pairing. When a primer "hybridizes" with a target (template), such a complex (or hybrid) is sufficiently stable to perform the priming function required, for example, by DNA polymerase to initiate DNA synthesis. It is understood that hybridizing sequences do not need to have perfect complementarity to provide a stable hybrid. In many cases, stable hybrids are formed with less than about 10% mismatch of bases, ignoring loops of 4 or more nucleotides. Thus, as used herein, the term "complementary" refers to an oligonucleotide that forms a stable duplex with its "complement" under assay conditions where there is generally about 90% or more homology.

[0048] SNAIL oligonucleotide primers In the subject method, the SNAIL oligonucleotide primer comprises at least a first oligonucleotide and a second oligonucleotide, each of the first oligonucleotide and the second oligonucleotide comprising a first region of complementarity, a second region of complementarity, and a third region of complementarity, the second oligonucleotide further comprising a barcode sequence, the first region of complementarity of the first oligonucleotide being complementary to a first portion of the target nucleic acid, the second region of complementarity of the first oligonucleotide being complementary to the first region of complementarity of the second oligonucleotide, the third region of complementarity of the first oligonucleotide being complementary to the third region of complementarity of the second oligonucleotide, the second region of complementarity of the second oligonucleotide being complementary to a second portion of the target nucleic acid, and the first region of complementarity of the first oligonucleotide being adjacent to the second region of complementarity of the second oligonucleotide. In an alternative embodiment, the second oligonucleotide is a closed circular molecule and the ligation step is omitted.

[0049] The present disclosure provides a method in which contacting fixed and permeabilized tissue comprises hybridizing a plurality of oligonucleotide primers having specificity for different target nucleic acids. In some embodiments, the method comprises a plurality of first oligonucleotides, including but not limited to, 5 or more first oligonucleotides hybridizing to target nucleotide sequences, such as 8 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more. In some embodiments, the method of the present disclosure comprises a plurality of first oligonucleotides, including but not limited to, 15 or more first oligonucleotides hybridizing to 15 or more, such as 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different target nucleotide sequences. In some embodiments, the method includes a plurality of second oligonucleotides, including but not limited to 5 or more second oligonucleotides, for example, 8 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more. In some embodiments, the method of the present disclosure includes a plurality of second oligonucleotides, including but not limited to 15 or more second oligonucleotides, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different first oligonucleotides that hybridize to 15 or more different target nucleotide sequences. Multiple oligonucleotide pairs can be used in the reaction, with one or more pairs specifically binding to each target nucleic acid. For example, two primer pairs can be used for one target nucleic acid to improve sensitivity and reduce variability.It is also of interest to detect multiple different target nucleic acids in a cell, for example, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 12, up to 15, up to 18, up to 20, up to 25, up to 30, up to 40 or more different target nucleic acids. Primers are typically denatured prior to use, typically by heating to a temperature of at least about 50°C, at least about 60°C, at least about 70°C, at least about 80°C, and up to about 99°C, up to about 95°C, up to about 90°C.

[0050] In some embodiments, the primers are denatured by heating prior to contacting the sample. In certain aspects, the melting temperature (T m ) is chosen to minimize ligation in solution. The "melting temperature" or "T m " is defined as the temperature at which half of the helical structure of a nucleic acid is lost due to heating or other dissociation of the hydrogen bonds between base pairs, e.g., by acid or alkali treatment. m depends on its length and base composition. Nucleic acid molecules rich in GC base pairs have a higher T m The complementary strands of the separated nucleic acids have a temperature T m When the temperature drops below T, the nucleic acid will spontaneously reassociate or anneal to form a double-stranded nucleic acid. m This occurs approximately 25°C lower than T m can be estimated using the following relationship: T m =69.3+0.41(GC)% (Marmur et al. (1962) J. Mol. Biol. 5:109-118).

[0051] In certain embodiments, the plurality of second oligonucleotides comprises a padlock probe. In some embodiments, the probe comprises a detectable label that can be measured and quantified. The terms "label" and "detectable label" refer to a molecule that can be detected, including, but not limited to, a radioisotope, a fluorescer, a chemiluminescer, an enzyme, an enzyme substrate, an enzyme cofactor, an enzyme inhibitor, a chromophore, a dye, a metal ion, a metal sol, a ligand (e.g., biotin or a hapten), and the like. The term "fluorescer" refers to a substance or a portion thereof that can exhibit fluorescence within the detectable range. Specific examples of labels that can be used with the present invention include, but are not limited to, phycoerythrin, Alexa dyes, fluorescein, YPet, CyPet, cascade blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas Red, luminol, acridinium esters, biotin, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), firefly luciferase, Renilla luciferase, NADPH, beta-galactosidase, horseradish peroxidase, glucose oxidase, alkaline phosphatase, chloramphenicol acetyltransferase, and urease.

[0052] In some embodiments, one or more of the first and second oligonucleotides bind to different regions of the target nucleic acid or target site. In a pair, each target site is different, and the target sites are adjacent sites on the target nucleic acid, e.g., typically no more than 15 nucleotides away from the other site, e.g., no more than 10, 8, 6, 4, or 2 nucleotides away, and may be contiguous sites. The target sites are typically on the same strand of the target nucleic acid in the same orientation. The target sites are also selected to provide unique binding sites for other nucleic acids present in the cell. Each target site is generally about 19 to about 25 nucleotides long, e.g., about 19 to 23 nucleotides, about 19 to 21 nucleotides, or about 19 to 20 nucleotides. The pair of first and second oligonucleotides is selected such that each oligonucleotide in the pair has a similar melting temperature for binding to its cognate target site, e.g., T m can be from about 50° C., from about 52° C., from about 55° C., from about 58° C., from about 62° C., from about 65° C., from about 70° C., or from about 72° C. The GC content of the target site is generally selected to be about 20% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less.

[0053] In some embodiments, the first oligonucleotide comprises a first, a second, and a third complementary region. The target site of the first oligonucleotide may refer to the first complementary region. As summarized above, the first complementary region of the first oligonucleotide may have a length of 19-25 nucleotides. In certain aspects, the second complementary region of the first oligonucleotide has a length of 3-10 nucleotides, including, for example, 4-8 nucleotides or 4-7 nucleotides. In some aspects, the second complementary region of the first oligonucleotide has a length of 6 nucleotides. In some embodiments, the third complementary region of the first oligonucleotide similarly has a length of 6 nucleotides. In such embodiments, the third complementary region of the first oligonucleotide has a length of 3-10 nucleotides, including, for example, 4-8 nucleotides or 4-7 nucleotides.

[0054] In some embodiments, the second first oligonucleotide comprises a first, second and third complementary region. The target site of the second oligonucleotide may refer to the second complementary region. As summarized above, the second complementary region of the second oligonucleotide may have a length of 19-25 nucleotides. In certain aspects, the first complementary region of the first oligonucleotide has a length of 3-10 nucleotides, including, for example, 4-8 nucleotides or 4-7 nucleotides. In some aspects, the first complementary region of the first oligonucleotide has a length of 6 nucleotides. In some aspects, the first complementary region of the second oligonucleotide comprises the 5' end of the second oligonucleotide. In some embodiments, the third complementary region of the second oligonucleotide similarly has a length of 6 nucleotides. In such embodiments, the third complementary region of the second oligonucleotide has a length of 3-10 nucleotides, including, for example, 4-8 nucleotides or 4-7 nucleotides. In further embodiments, the third region of complementarity of the second oligonucleotide comprises the 3' end of the second oligonucleotide. In some embodiments, the first region of complementarity of the second oligonucleotide is adjacent to the third region of complementarity of the second oligonucleotide.

[0055] In some embodiments, the second oligonucleotide comprises a barcode sequence, and the barcode sequence of the second oligonucleotide provides barcoded information for identifying the target nucleic acid. The term "barcode" refers to a nucleic acid sequence used to identify a single cell or a subpopulation of cells. The barcode sequence can be linked to the target nucleic acid of interest during amplification and can be used to trace the amplicon to the cell from which the target nucleic acid originated. The barcode sequence can be added to the target nucleic acid of interest during amplification by performing the amplification with an oligonucleotide that contains a region that includes the barcode sequence and a region that is complementary to the target nucleic acid such that the barcode sequence is ultimately incorporated into the amplified target nucleic acid product (i.e., amplicon).

[0056] In some embodiments, the first oligonucleotide contains a unique matching sequence (SNAIL matching sequence) that is used to complement each end of the second oligonucleotide (a SNAIL shell that hybridizes the ends together and is then ligated). In some embodiments, the second complementary region of the first oligonucleotide contains a first portion of the unique matching sequence, the third complementary region of the first oligonucleotide contains a second portion of the unique matching sequence, the first complementary region of the second oligonucleotide contains a sequence that is complementary to the first portion of the unique matching sequence, and the third complementary region of the second oligonucleotide contains a sequence that is complementary to the second portion of the unique matching sequence. The unique matching sequence minimizes spurious probe-probe interactions that may arise due to either off-target hybridization, protein binding, or other probe aggregation or persistent close interactions, improving specificity when a greater number of genes are targeted or higher probe concentrations are used. The method relies on the specificity of the ligase, and a ligase that does not tolerate mismatched sequences is used. To detect multiple different nucleic acid targets, a unique matching sequence per gene or per probe pair can be generated.

[0057] In some embodiments, the first oligonucleotide further comprises a common binding site for the gel adaptor oligonucleotide. The gel adaptor oligonucleotide comprises a functional linkage modification, such as an acrylidine, at its 5' or 3' end, such that the first oligonucleotide is covalently linked to the hydrogel via the gel adaptor oligonucleotide during gelation. The use of the gel adaptor helps to retain the amplicon grown from the 3' end of the first oligonucleotide in the gel, without requiring the first oligonucleotide to have a 5' modification itself. In some embodiments, the common binding site for the gel adaptor oligonucleotide is adjacent to the first complementary region of the first oligonucleotide.

[0058] In certain embodiments, mRNA-holding oligonucleotides are used to hold the mRNA in the hydrogel during or before hybridization. The mRNA-holding oligonucleotides contain a nucleotide modification, such as an acrydite modification at the 5' or 3' end, which allows the mRNA-holding oligonucleotide to be linked to the hydrogel during gelation, a poly-T tail that hybridizes to the poly-A tail of the mRNA, such that hybridization of the poly-T tail of the mRNA-holding oligonucleotide with the poly-A tail of the mRNA holds the mRNA in the hydrogel, and a unique hybridization sequence. In some embodiments, the poly-T tail contains reciprocal locked nucleic acid (LNA) thymine (T) bases. The use of the mRNA-holding oligonucleotide allows gelation to be performed prior to hybridization, if necessary. The unique hybridization sequence on the mRNA-holding oligonucleotide allows for labeling of the mRNA by hybridization with a complementary fluorescently labeled oligonucleotide probe.

[0059] organization As described herein, the disclosed methods include in situ sequencing techniques by contacting fixed and permeabilized intact tissue with at least one pair of oligonucleotide primers under conditions that allow specific hybridization. Tissue specimens suitable for use with the methods described herein generally include any type of tissue specimen collected from a living or cadaveric subject, such as, for example, epithelial, muscle, connective tissue, and neural tissue, including but not limited to, those biopsy and autopsy specimens. The tissue specimen may be collected and processed using the methods described herein and subjected to microscopic analysis immediately after processing, or may be preserved and subjected to microscopic analysis in the future, for example after long-term storage. In some embodiments, the methods described herein may be used to preserve the tissue specimen in a stable, accessible, and completely intact form for future analysis. In some embodiments, the methods described herein may be used to analyze previously preserved or archived tissue specimens. In some embodiments, the intact tissue includes brain tissue, such as visual cortex slices. In some embodiments, the intact tissue is a thin slice having a thickness of 5-20 μm, for example, but not limited to, 5-18 μm, 5-15 μm, or 5-10 μm, while in other embodiments, the intact tissue is a thick slice having a thickness of 50-200 μm, for example, but not limited to, 50-150 μm, 50-100 μm, or 50-80 μm.

[0060] An embodiment of the present invention includes fixing intact tissue. The term "fixing" or "fixation" as used herein is a process of preserving biological material (e.g., tissues, cells, organelles, molecules, etc.) from decay and / or decomposition. Fixation can be accomplished using any convenient protocol. Fixation can include contacting the sample with a fixation reagent (i.e., a reagent containing at least one fixation agent). The sample can be contacted with the fixation reagent for a wide range of times that can depend on the temperature, the nature of the sample, and the fixation agent. For example, the sample can be contacted with the fixation reagent for 24 hours or less, 18 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2 minutes or less.

[0061] The sample can be contacted with the immobilization reagent for a period ranging from 5 minutes to 24 hours, e.g., 10 minutes to 20 hours, 10 minutes to 18 hours, 10 minutes to 12 hours, 10 minutes to 8 hours, 10 minutes to 6 hours, 10 minutes to 4 hours, 10 minutes to 2 hours, 15 minutes to 20 hours, 15 minutes to 18 hours, 15 minutes to 12 hours, 15 minutes to 8 hours, 15 minutes to 6 hours, 15 minutes to 4 hours, 15 minutes to 2 hours, 15 minutes to 1.5 hours, 15 minutes to 1 hour, 10 minutes to 30 minutes, 15 minutes to 30 minutes, 30 minutes to 2 hours, 45 minutes to 1.5 hours, or 55 minutes to 70 minutes.

[0062] The sample may be contacted by the immobilization reagent at a variety of temperatures, depending on the protocol and reagents used. For example, in some cases, the sample may be contacted by the immobilization reagent at a temperature ranging from -22°C to 55°C, with particular ranges of interest including, but not limited to, 50-54°C, 40-44°C, 35-39°C, 28-32°C, 20-26°C, 0-6°C, and -18-22°C. In some cases, the sample may be contacted by the immobilization reagent at temperatures of -20°C, 4°C, room temperature (22-25°C), 30°C, 37°C, 42°C, or 52°C.

[0063] Any convenient fixation reagent may be used. Common fixation reagents include cross-linking fixation agents, precipitation fixation agents, oxidation fixation agents, mercury, and the like. Cross-linking fixation agents chemically join two or more molecules by covalent bonds, and a wide range of cross-linking reagents may be used. Examples of suitable cross-linking fixation agents include, but are not limited to, aldehydes (e.g., formaldehyde, commonly also referred to as "paraformaldehyde" and "formalin", glutaraldehyde, and the like), imidoesters, NHS (N-hydroxysuccinimide) esters, and the like. Examples of suitable precipitation fixation agents include, but are not limited to, alcohols (e.g., methanol, ethanol, and the like), acetone, acetic acid, and the like. In some embodiments, the fixation agent is formaldehyde (i.e., paraformaldehyde or formalin). Suitable final concentrations of formaldehyde in the fixation reagent are 0.1-10%, 1-8%, 1-4%, 1-2%, 3-5%, or 3.5-4.5%, including about 1.6%, for 10 minutes. In some embodiments, the sample is fixed at a final concentration of 4% formaldehyde (diluted from a more concentrated stock solution, e.g., 38%, 37%, 36%, 20%, 18%, 16%, 14%, 10%, 8%, 6%, etc.). In some embodiments, the sample is fixed at a final concentration of 10% formaldehyde. In some embodiments, the sample is fixed at a final concentration of 1% formaldehyde. In some embodiments, the fixative is glutaraldehyde. Suitable concentrations of glutaraldehyde in the fixation reagent are 0.1-1%. The fixation reagent may contain two or more fixatives in any combination. For example, in some embodiments, the sample is contacted with a fixation reagent that contains both formaldehyde and glutaraldehyde.

[0064] The term "permeabilization" or "permeabilize" as used herein refers to a process of making cells (e.g., cell membranes) of a sample permeable to experimental reagents such as nucleic acid probes, antibodies, chemical substrates, etc. Any convenient method and / or reagent for permeabilization may be used. Suitable permeabilization reagents include detergents (e.g., saponin, Triton X-100, Tween-20, etc.), organic fixatives (e.g., acetone, methanol, ethanol, etc.), enzymes, etc. Detergents may be used in a wide range of concentrations. For example, 0.001% to 1% detergent, 0.05% to 0.5% detergent, or 0.1% to 0.3% detergent may be used for permeabilization (e.g., 0.1% saponin, 0.2% tween-20, 0.1 to 0.3% Triton X-100, etc.). In some embodiments, permeabilization is performed using methanol on ice for at least 10 minutes.

[0065] In some embodiments, the same solution may be used as the fixation reagent and the permeabilization reagent. For example, in some embodiments, the fixation reagent contains 0.1%-10% formaldehyde and 0.001%-1% saponin. In some embodiments, the fixation reagent contains 1% formaldehyde and 0.3% saponin.

[0066] The sample can be contacted with the permeabilization reagent for a wide range of times, which can depend on the temperature, the nature of the sample, and the permeabilization reagent. For example, the sample can be contacted with the permeabilization reagent for 24 hours or more, 24 hours or less, 18 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2 minutes or less. The sample can be contacted with the permeabilization reagent at a variety of temperatures, depending on the protocol and reagents used. For example, in some examples, the sample can be contacted with the permeabilization reagent at a temperature ranging from -82°C to 55°C, with particular ranges of interest including 50-54°C, 40-44°C, 35-39°C, 28-32°C, 20-26°C, 0-6°C, -18-22°C, and -78-82°C. In some cases, the sample can be contacted by the permeabilization reagent at a temperature of -80°C, -20°C, 4°C, room temperature (22-25°C), 30°C, 37°C, 42°C, or 52°C.

[0067] In some embodiments, the sample is contacted with an enzyme permeabilization reagent. The enzyme permeabilization reagent permeabilizes the sample by partially degrading extracellular matrix or surface proteins that prevent permeabilization of the sample by the assay reagents. Contact with the enzyme permeabilization reagent can occur at any time after fixation and before target detection. In some cases, the enzyme permeabilization reagent is proteinase K, a commercially available enzyme. In such cases, the sample is contacted with proteinase K before contacting with the post-fixation reagent. Proteinase K treatment (i.e., contact with proteinase K, also commonly referred to as "proteinase K digestion") can be performed over a range of times, at a range of temperatures, and over a range of enzyme concentrations that are empirically determined for each cell or tissue type under investigation. For example, the sample can be contacted with proteinase K for 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2 minutes or less. The sample can be contacted with Proteinase K at 1 μg / ml or less, 2 μg / ml or less, 4 μg / ml or less, 8 μg / ml or less, 10 μg / ml or less, 20 μg / ml or less, 30 μg / ml or less, 50 μg / ml or less, or 100 μg / ml or less. The sample can be contacted with Proteinase K at a temperature ranging from 2° C. to 55° C., with specific ranges of interest including, but not limited to, 50-54° C., 40-44° C., 35-39° C., 28-32° C., 20-26° C., and 0-6° C. In some cases, the sample can be contacted with Proteinase K at a temperature of 4° C., room temperature (22-25° C.), 30° C., 37° C., 42° C., or 52° C. In some embodiments, the sample is not contacted with an enzyme permeabilization reagent. In some embodiments, the sample is not contacted with Proteinase K. Contacting the intact tissue with at least a fixation reagent and a permeabilization reagent results in the production of fixed and permeabilized tissue.

[0068] Ligase In some embodiments, the disclosed method comprises adding a ligase to ligate the second oligonucleotide and generate a closed nucleic acid circle. In some embodiments, the addition of the ligase comprises the addition of a DNA ligase. In alternative embodiments, the second oligonucleotide is provided as a closed nucleic acid circle, and the step of adding the ligase is omitted. In certain embodiments, the ligase is an enzyme that facilitates sequencing of the target nucleic acid molecule.

[0069] The term "ligase" as used herein refers to an enzyme that is commonly used to join polynucleotides together or to join the ends of a single polynucleotide. Ligases include ATP-dependent double-stranded polynucleotide ligases, NAD-i-dependent double-stranded DNA or RNA ligases, and single-stranded polynucleotide ligases, such as any of the ligases described in EC 6.5.1.1 (ATP-dependent ligases), EC 6.5.1.2 (NAD+-dependent ligases), EC 6.5.1.3 (RNA ligases). Specific examples of ligases include bacterial ligases such as E. coli DNA ligase and Taq DNA ligase, Ampligase® thermostable DNA ligase (Epicentre® Technologies Corp., part of Illumina®, Madison, Wis.), and phage ligases such as T3 DNA ligase, T4 DNA ligase, and T7 DNA ligase, and variants thereof.

[0070] Rolling Circle Amplification In some embodiments, the method of the present invention comprises performing rolling circle amplification in the presence of a nucleic acid molecule, which comprises forming one or more amplicons using a second oligonucleotide as a template and a first oligonucleotide as a primer for a polymerase. In such an embodiment, a single-stranded circular polynucleotide template is formed by ligation of the second oligonucleotide, and the circular polynucleotide comprises a region that is complementary to the first oligonucleotide. Upon addition of DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, the first oligonucleotide is extended by replication of multiple copies of the template. This amplification product can be easily detected by binding to a detection probe. In some embodiments, the polymerase is pre-incubated without dNTPs to allow the polymerase to uniformly permeate the sample before performing rolling circle amplification.

[0071] In some embodiments, only if the first and second oligonucleotides hybridize to the same target nucleic acid molecule, the second oligonucleotide may be circularized and rolled circle amplified to generate cDNA nanoballs (i.e., amplicons) containing multiple copies of the cDNA. The term "amplicon" refers to the amplified nucleic acid product of a PCR reaction or other nucleic acid amplification process. In some embodiments, amine-modified nucleotides are spiked into the rolling circle amplification reaction.

[0072] Techniques for rolling circle amplification are known in the art (e.g., Baner et al, Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al, Nature Genetics 19:226, 1998; Schweitzer et al. Proc. Natl Acad. Sci. USA 97:101 13-1 19, 2000; Faruqi et al, BMC Genomics 2:4, 2000; Nallur et al, Nucl. Acids Res. 29:el18, 2001; Dean et al. Genome Res. 11:1095-1099, 2001; Schweitzer et al, Nature (See, Biotech. 20:359-365, 2002; U.S. Patent Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329, and 6,368,801.) In some embodiments, the polymerase is a phi29 DNA polymerase.

[0073] In certain aspects, the nucleic acid molecule comprises an amine-modified nucleotide. In such an embodiment, the amine-modified nucleotide comprises an acrylate N-hydroxysuccinimide partial modification. Other examples of amine-modified nucleotides include, but are not limited to, a 5-aminoallyl-dUTP partial modification, a 5-propargylamino-dCTP partial modification, an N6-6-aminohexyl-dATP partial modification, or a 7-deaza-7-propargylamino-dATP partial modification.

[0074] In some embodiments, rolling circle amplification is carried out in the presence of oligonucleotides that label and condense the amplicons ("amplicon condensation and detection oligonucleotides"). The amplicon condensation and detection oligonucleotides contain terminal modifications, such as 5' primary amines or acrydite modifications, for binding to a gel. In addition, the amplicon condensation and detection oligonucleotides contain a unique sequence for hybridization of a probe for amplicon detection and two or more copies of a sequence complementary to a common sequence on the amplicon, so that the amplicon condensation and detection oligonucleotides pull the strands together from a given amplicon. In some embodiments, the amplicon condensation and detection oligonucleotides contain three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more copies of a sequence complementary to a common sequence on the amplicon. The use of amplicon condensation and detection oligonucleotides reduces the optical spread of the amplicons, making adjacent amplicons from different genes less likely to aggregate when closely packed. In some embodiments, amplicon condensation and detection oligonucleotides are used for in situ gene sequencing of target nucleic acids in thick tissue sections.

[0075] Amplicon embedding in tissue-hydrogel setup In some embodiments, the disclosed methods include embedding one or more amplicons in the presence of hydrogel subunits to form one or more hydrogel-embedded amplicons. The described hydrogel histochemistry involves covalently attaching nucleic acids to in situ synthesized hydrogels for tissue clearing, enzyme diffusion, and multiple cycle sequencing, which existing hydrogel histochemistry methods are unable to do. In some embodiments, to enable amplicon embedding in a tissue-hydrogel setup, amine-modified nucleotides are spiked into a rolling circle amplification reaction, functionalized with acrylamide moieties using acrylic acid N-hydroxysuccinimide ester, and copolymerized with acrylamide monomers to form a hydrogel.

[0076] As used herein, the term "hydrogel" or "hydrogel network" refers to a network of water-insoluble polymer chains that are sometimes found as colloidal gels in which water is the dispersion medium. In other words, hydrogels are a class of polymeric materials that can absorb large amounts of water without dissolving. Hydrogels can contain more than 99% water and can include natural or synthetic polymers, or combinations thereof. Hydrogels also have a degree of flexibility that is very similar to natural tissues due to their significant water content. A detailed description of suitable hydrogels can be found in U.S. Patent Application Publication No. 2010 / 0055733, which is specifically incorporated herein by reference. As used herein, the term "hydrogel subunit" or "hydrogel precursor" refers to hydrophilic monomers, prepolymers, or polymers that can be crosslinked or "polymerized" to form a three-dimensional (3D) hydrogel network. Without being bound by any scientific theory, it is believed that this fixation of the biological specimen in the presence of the hydrogel subunits crosslinks components of the specimen to the hydrogel subunits, thereby fixing the molecular components in place and preserving tissue architecture and cellular morphology.

[0077] In some embodiments, embedding comprises copolymerizing one or more amplicons with acrylamide. As used herein, the term "copolymer" describes a polymer that contains two or more types of subunits. The term encompasses polymers that contain two, three, four, five, or six types of subunits.

[0078] In certain aspects, embedding comprises clarification of the one or more hydrogel-embedded amplicons, and the target nucleic acid is substantially retained in the one or more hydrogel-embedded amplicons. In such embodiments, clarification comprises substantially removing a plurality of cellular components from the one or more hydrogel-embedded amplicons. In some other embodiments, clarifying comprises substantially removing lipids and / or proteins from the one or more hydrogel-embedded amplicons. As used herein, the term "substantially" means that the original amount present in the sample prior to clarification is reduced by approximately 70% or more, such as 75% or more, such as 80% or more, such as 85% or more, such as 90% or more, such as 95% or more, such as 99% or more, such as 100%.

[0079] In some embodiments, clarifying the amplicons embedded in the hydrogel comprises performing electrophoresis on the specimen. In some embodiments, the amplicons are electrophoresed using a buffer solution comprising an ionic detergent. In some embodiments, the ionic detergent is sodium dodecyl sulfate (SDS). In some embodiments, the specimen is electrophoresed using a voltage ranging from about 10 to about 60 volts. In some embodiments, the specimen is electrophoresed for a period ranging from about 15 minutes up to about 10 days. In some embodiments, the method further comprises incubating the cleared specimen in an encapsulation medium having a refractive index that matches the refractive index of the cleared tissue. In some embodiments, the encapsulation medium increases the optical clarity of the specimen. In some embodiments, the encapsulation medium comprises glycerol.

[0080] Ligation sequencing of SCAL and SEDAL2 In some embodiments, SEDAL2 or SCAL sequencing by ligation is used. The methods disclosed herein include contacting one or more hydrogel-embedded amplicons having a barcode sequence with a pair of primers under conditions that allow ligation, the pair of primers including a third oligonucleotide and a fourth oligonucleotide, and ligation occurs only when both the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon. In some embodiments, the third oligonucleotide is configured to decode a base, and the fourth oligonucleotide is configured to convert the decoded base into a signal. In some aspects, the signal is a fluorescent signal. In an exemplary aspect, contacting one or more hydrogel-embedded amplicons having a barcode sequence with a pair of primers under conditions that allow ligation includes ligation of each of the third oligonucleotide and the fourth oligonucleotide to form a stable product for imaging only if a perfect match occurs. In certain aspects, the mismatch sensitivity of a ligase enzyme is used to determine the underlying sequence of a target nucleic acid molecule.

[0081] The inclusion of a polyethylene glycol (PEG) polymer in the sequencing ligation mixture substantially accelerates signal attachment to the target nucleic acid. Exemplary PEG polymers have molecular weights ranging from 300 g / mol to 10,000,000 g / mol. In some embodiments, a PEG 6000 polymer is present during ligation of the third and fourth oligonucleotides.

[0082] In some embodiments, the contacting of the one or more hydrogel-embedded amplicons occurs two or more times, including, but not limited to, for example, three or more, four or more, five or more, six or more, or seven or more times. In certain embodiments, the contacting of the one or more hydrogel-embedded amplicons occurs four or more times for thin tissue specimens. In other embodiments, the contacting of the one or more hydrogel-embedded amplicons occurs six or more times for thick tissue specimens. In some embodiments, the one or more amplicons may be contacted with a pair of primers for 24 hours or more, 24 hours or less, 18 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 2 minutes or less. In some embodiments, the method is performed at room temperature for preservation of tissue morphology and has low background noise and error reduction. In some embodiments, contacting the one or more hydrogel-embedded amplicons comprises eliminating the accumulation of errors as sequencing progresses.

[0083] Specimens prepared using the present methods can be analyzed by any of several different types of microscopy, including optical microscopy (e.g., bright field, oblique illumination, dark field, phase contrast, differential interference contrast, interference reflection, epifluorescence, confocal, etc.), laser microscopy, electron microscopy, and scanning probe microscopy. In some embodiments, the non-transitory computer readable medium converts the raw images acquired through multiple rounds of in situ sequencing microscopy into decoded gene identities and spatial locations, and then analyzes the per-cell composition of gene expression.

[0084] The term "perfectly matched", when used in reference to a double strand, means that the polynucleotides and / or oligonucleotide strands constituting the double strand form a double-stranded structure with each other, such that every nucleotide in each strand undergoes Watson-Crick base pairing with the nucleotide in the other strand. The term "double strand" includes, but is not limited to, pairings of nucleoside analogs such as deoxyinosine, nucleosides with 2-aminopurine bases, peptide nucleic acids (PNAs), etc., may be used. A "mismatch" in a double strand between two oligonucleotides means that a pair of nucleotides in the double strand cannot undergo Watson-Crick binding.

[0085] In some embodiments, the methods include a plurality of third oligonucleotides, including but not limited to, 5 or more third oligonucleotides, e.g., 8 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more, that hybridize to a target nucleotide sequence. In some embodiments, the methods of the disclosure include a plurality of third oligonucleotides, including but not limited to, 15 or more third oligonucleotides, e.g., 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different target nucleotide sequences, that hybridize to a target nucleotide sequence. In some embodiments, the method includes a plurality of fourth oligonucleotides, including but not limited to 5 or more fourth oligonucleotides, for example, 8 or more, 10 or more, 12 or more, 15 or more, 18 or more, 20 or more, 25 or more, 30 or more, 35 or more. In some embodiments, the method of the present disclosure includes a plurality of fourth oligonucleotides, including but not limited to 15 or more fourth oligonucleotides, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different first oligonucleotides, hybridizing to 15 or more, for example, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, and up to 80 different target nucleotide sequences. Multiple oligonucleotide pairs can be used in the reaction, with one or more pairs specifically binding to each target nucleic acid. For example, two primer pairs can be used for one target nucleic acid to improve sensitivity and reduce variability. It is also of interest to detect multiple different target nucleic acids within a cell, for example, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 12, up to 15, up to 18, up to 20, up to 25, up to 30, up to 40 or more different target nucleic acids.

[0086] In certain embodiments, sequencing is performed with a ligase whose activity is hindered by the base mismatch, the third oligonucleotide, and the fourth oligonucleotide. The term "hindered" in this context refers to the activity of the ligase being reduced by approximately 20% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more, such as 99% or more, such as 100%. In some embodiments, the third oligonucleotide has a length of 5-15 nucleotides, including but not limited to 5-13 nucleotides, 5-10 nucleotides, or 5-8 nucleotides. In some embodiments, the T of the third oligonucleotide is m is room temperature (22-25° C.). In some embodiments, the third oligonucleotide is denatured or partially denatured. In some embodiments, the fourth oligonucleotide has a length of 5-15 nucleotides, including but not limited to 5-13 nucleotides, 5-10 nucleotides, or 5-8 nucleotides. In some embodiments, the T of the fourth oligonucleotide is m is room temperature (22-25°C). After each cycle of sequencing corresponding to a base readout, the fourth oligonucleotide may be stripped off, which eliminates error accumulation as sequencing proceeds. In some embodiments, the fourth oligonucleotide is stripped off by formamide.

[0087] In some embodiments, sequencing involves washing the third and fourth oligonucleotides to remove unbound oligonucleotides, and then revealing the fluorescent product for imaging. In certain exemplary embodiments, detectable labels may be used to detect one or more nucleotides and / or oligonucleotides described herein. In certain embodiments, detectable labels may be used to detect one or more amplicons. Examples of detectable markers include various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, protein-protein binding pairs, protein-antibody binding pairs, and the like. Examples of fluorescent proteins include, but are not limited to, yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, and the like. Examples of bioluminescent markers include, but are not limited to, luciferase (e.g., bacterial, firefly, click beetle, and the like), luciferin, aequorin, and the like. Examples of enzyme systems with visually detectable signals include, but are not limited to, galactosidase, glucolimidase, phosphatase, peroxidase, cholinesterase, etc. Identifiable markers include: 125 I, 35 S, 14 C, or 3 Also included are radioactive compounds such as 3H. Identifiable markers are commercially available from a variety of sources.

[0088] Fluorescent labels and their attachment to nucleotides and / or oligonucleotides are described in many publications, including Haugland, Handbook of Fluorescent Probes and Research Chemicals, 9th Edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993); Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); and Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991). Specific methodologies applicable to the present invention are disclosed in the example references in U.S. Patent Nos. 4,757,141, 5,151,507, and 5,091,519. In one embodiment, one or more fluorescent dyes are used as labels for labeled target sequences, e.g., as disclosed by U.S. Pat. No. 5,188,934 (4,7-dichlorofluorescein dyes), U.S. Pat. No. 5,366,860 (spectrally resolvable rhodamine dyes), U.S. Pat. No. 5,847,162 (4,7-dichlororhodamine dyes), U.S. Pat. No. 4,318,846 (ether-substituted fluorescein dyes), U.S. Pat. No. 5,800,996 (energy transfer dyes), Lee et al., U.S. Pat. No. 5,066,580 (xanthine dyes), U.S. Pat. No. 5,688,648 (energy transfer dyes), and the like. Labeling can be done with quantum dots, as disclosed in the following patents and patent publications: U.S. Patent Nos. 6,322,901, 6,576,291, 6,423,551, 6,251,303, 6,319,426, 6,426,513, 6,444,143, 5,990,479, 6,207,392, 2002 / 0045045, and 2003 / 0017264.As used herein, the term "fluorescent label" includes signaling moieties that transmit information through the fluorescent absorption and / or emission properties of one or more molecules. Such fluorescent properties include fluorescence intensity, fluorescence lifetime, emission spectral properties, energy transfer, and the like.

[0089] Commercially available fluorescent nucleotide analogs that are readily incorporated into nucleotide and / or oligonucleotide sequences include Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, NJ), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHODAMINE GREEN™-5-dUTP, OREGON GREEN™ 488-5 ...5-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-dUTP, TEXAS RED™-5-d RED(TM)-12-dUTP, BODIPY(TM) 630 / 650-14-dUTP, BODIPY(TM) 650 / 665-14-dUTP, ALEXA FLUOR(TM) 488-5-dUTP, ALEXA FLUOR(TM) 532-5-dUTP, ALEXA FLUOR(TM) 568-5-dUTP, ALEXA FLUOR(TM) 594-5-dUTP, ALEXA FLUOR(TM) 546-14-dUTP, Fluorescein-12-UTP, Tetramethylrhodamine-6-UTP, TEXAS RED(TM)-5-UTP, mCherry, CASCADE BLUE(TM)-7-UTP, BODIPY(TM) FL-14-UTP, BODIPY These include, but are not limited to, TMR-14-UTP, BODIPY™ TR-14-UTP, RHODAMINE GREEN™-5-UTP, ALEXA FLUOR™ 488-5-UTP, LEXA FLUOR™ 546-14-UTP (Molecular Probes, Inc. Eugene, Oreg.), etc. Protocols for the custom synthesis of nucleotides bearing other fluorophores are known in the art (see Henegariu et al. (2000) Nature Biotechnol. 18:345).

[0090] Other fluorophores available for post-synthetic conjugation include ALEXA FLUOR™ 350, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 647, BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY Examples of fluorophores include, but are not limited to, 650 / 665, Cascade Blue, Cascade Yellow, Dansyl, Lissamine Rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, Oreg.), Cy2, Cy3.5, Cy5.5, Cy7 (Amersham Biosciences, Piscataway, NJ), etc. FRET tandem fluorophores can also be used, including, but are not limited to, PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680), APC-Alexa dyes, etc.

[0091] Metallic silver or gold particles can be used to enhance the signal from fluorescently labeled nucleotide and / or oligonucleotide sequences (Lakowicz et al. (2003) BioTechniques 34:62).

[0092] Biotin or its derivatives can also be used as a label on the nucleotide and / or oligonucleotide sequence, which can then be bound by detectably labeled avidin / streptavidin derivatives (e.g., phycoerythrin-conjugated streptavidin) or detectably labeled anti-biotin antibodies. Digoxigenin can be incorporated as a label, which can then be bound by detectably labeled anti-digoxigenin antibodies (e.g., fluoresceinated anti-digoxigenin). Aminoallyl-dUTP residues can be incorporated into the oligonucleotide sequence, which can then be bound to N-hydroxysuccinimide (NHS)-derivatized fluorescent dyes. In general, any member of the conjugate pair can be incorporated into the detection oligonucleotide, provided that the detectably labeled conjugate partner can be bound in a manner that allows detection. As used herein, the term antibody refers to any class of antibody molecule, or any subfragment thereof, such as Fab.

[0093] Other suitable labels for oligonucleotide sequences may include fluorescein (FAM), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis), phosphorescent amino acids (e.g., P-tyr, P-ser, P-thr), etc. In one embodiment, the following hapten / antibody pairs are used for detection, with each of the antibodies derivatized with a detectable label: biotin / α-biotin, digoxigenin / α-digoxigenin, dinitrophenol (DNP) / α-DNP, 5-carboxyfluorescein (FAM) / α-FAM.

[0094] In certain exemplary embodiments, the nucleotide and / or oligonucleotide sequences may be indirectly labeled with a hapten that is then specifically bound by a capture agent, as disclosed, for example, in U.S. Pat. Nos. 5,344,757, 5,702,888, 5,354,657, 5,198,537, and 4,849,336, PCT Publication No. 91 / 17160, and the like. Many different hapten capture agent pairs can be used. Exemplary haptens include, but are not limited to, biotin, desbiotin and other derivatives, dinitrophenol, dansyl, fluorescein, CY5, digoxigenin, and the like. In the case of biotin, the capture agent may be avidin, streptavidin, or an antibody. Antibodies can be used as capture agents for other haptens (many dye-antibody pairs are commercially available, e.g., Molecular Probes, Eugene, Oreg.).

[0095] In some embodiments, antioxidant compounds are included in the washing and imaging buffers (i.e., "anti-bleaching buffers") to reduce photobleaching during fluorescence imaging. Exemplary antioxidants include, but are not limited to, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and Trolox-quinone, propyl gallate, tertiary butyl hydroquinone, butylated hydroxyanisole, butylated hydroxytoluene, glutathione, ascorbic acid, and tocopherol. Such antioxidants have an anti-bleaching effect on fluorophores. That is, antioxidants reduce photobleaching during tiling and greatly enhance the signal-to-noise ratio (SNR) of sensitive fluorophores, allowing higher SNR imaging of thicker samples. For a fixed exposure time with antioxidants, the SNR is increased by increasing the concentration of fluorophores that are not bleached during exposure to light. The inclusion of antioxidants also removes the diminishing returns of longer exposure times (caused by limited fluorophore lifetime before photobleaching), providing an increase in SNR by allowing increased exposure times.

[0096] In addition, when multiple sequencing cycles are used, fluorophore cleavage or probe stripping from the probe can be used to eliminate signal carryover from one round to the next. For example, the fluorophore can be stripped away with formamide. Alternatively, thiol-linked dyes with a disulfide linkage between the fluorophore and the oligonucleotide probe can be used, which allows for cleavage of the fluorophore from the oligonucleotide probe under a reducing environment. Exemplary disulfide reducing agents that can be used to cleave disulfide bonds include, but are not limited to, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), and b-mercaptoethanol (BME). After fluorescent imaging during a sequencing round, a stripping agent and / or reducing agent is added, followed by a washing step to remove diffusible fluorescent signal before performing another round of sequencing.

[0097] A sequencing cycle for SCAL or SEDAL2 optionally begins with a short sample wash before proceeding to the first signal addition. For SCAL sequencing, depending on whether sequential or combinatorial encoding is used for a particular round, a corresponding set of third and fourth oligonucleotides and their round-specific competitors are added and ligated. In combinatorial encoding, a third oligonucleotide for a given position x is added, plus a set of fluorescently labeled dibase encoding oligonucleotides, plus a competitor oligonucleotide for the previous position that was labeled (unless it is the first round of labeling, in which case the competitor oligonucleotide is omitted). In sequential encoding, a third oligonucleotide for a given round x, a four-channel fluorophore mixture, and a round x-1 competitor oligonucleotide are added, unless it is the first round of labeling. The presence of PEG in the sequencing ligation mixture substantially accelerates signal addition to the target. After incubation of the sample in the imaging buffer, the sample is imaged and briefly rinsed before proceeding to the next sequencing cycle.

[0098] In the case of SEDAL2, the same oligonucleotide / ligation mixture as above is used during the signal addition step, except that the competitor oligonucleotide is omitted. After sample addition, washing, imaging buffer addition, and imaging as above, SEDAL2 includes a separate step for signal removal, where the signal is either stripped away with a formamide-containing stripping solution, or, if thiol-linked dyes are used to sequentially encode fluorescently labeled oligonucleotides, with a cleavage solution containing a disulfide reducing agent (e.g., TCEP). The sample is then washed before proceeding to the next round of signal addition.

[0099] cell The methods disclosed herein include methods for in situ genetic sequencing of target nucleic acids in cells in intact tissues. In certain embodiments, the cells are present in a population of cells. In certain other embodiments, the population of cells includes multiple cell types, including, but not limited to, excitatory neurons, inhibitory neurons, and non-neuronal cells. The cells used in the assays of the present invention can be organisms, single cell types from organisms, or mixtures of cell types. Included are naturally occurring cells and cell populations, genetically engineered cell lines, cells from transgenic animals, and the like. Virtually any cell type and size can be accommodated. Suitable cells include bacterial, fungal, plant, and animal cells. In one embodiment of the present invention, the cells are mammalian cells, such as naturally occurring tissues, complex cell populations, such as blood, liver, pancreas, neural tissue, bone marrow, skin, and the like. Some tissues can be disrupted into monodisperse suspensions. Alternatively, the cells can be cultured populations, such as cultures derived from complex populations, cultures derived from a single cell type in which the cells have differentiated into multiple lineages, or cells are differentially responsive to stimuli, and the like.

[0100] Cell types that may find use in the subject invention include stem and progenitor cells, e.g., embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, neural crest cells, etc., endothelial cells, muscle cells, cardiac muscle, smooth and skeletal muscle cells, mesenchymal cells, epithelial cells; hematopoietic cells, such as lymphocytes, including T cells, such as Th1 T cells, Th2 T cells, ThO T cells, cytotoxic T cells, etc.; B cells, pre-B cells, etc.; monocytes; dendritic cells; neutrophils; and macrophages; natural killer cells; mast cells, etc.; adipocytes, cells involved in specific organs, such as the thymus, endocrine glands, pancreas, brain, such as neurons, glia, astrocytes, dendritic cells, and genetically modified versions thereof. Hematopoietic cells may be associated with inflammatory processes, autoimmune diseases, etc., while endothelial cells, smooth muscle cells, cardiac muscle cells, etc. may be associated with cardiovascular diseases. Almost any type of cell may be associated with tumors, such as sarcomas, carcinomas, and lymphomas, liver diseases involving hepatocytes, kidney diseases involving renal cells, etc.

[0101] The cells may also be transformed or tumor cells of different types, e.g., carcinomas of different cellular origin, lymphomas of different cell types, etc. The American Type Culture Collection (Manassas, VA) has collected and made available over 4,000 cell lines from over 150 different species, over 950 cancer cell lines, including 700 human cancer cell lines. The National Cancer Institute has accumulated clinical, biochemical, and molecular data from a large panel of human tumor cell lines, which are available from the ATCC or NCI (Phelps et al. (1996) Journal of Cellular Biochemistry Supplement 24:32-91). Different cell lines that have been spontaneously derived or selected for desired growth or response characteristics from individual cell lines may include multiple cell lines derived from similar tumor types, but from different patients or sites.

[0102] The cells may be non-adherent, e.g., blood cells including monocytes, T cells, B cells, tumor cells, etc., or adherent cells, e.g., epithelial cells, endothelial cells, neuronal cells, etc. To profile adherent cells, they may be dissociated from the substrate to which they are adhered and from other cells in a manner that maintains the ability to recognize and bind to the probe molecule.

[0103] Such cells can be obtained from an individual, for example, by a variety of techniques known in the art, from a variety of tissues, e.g., blood, bone marrow, solid tissues (e.g., solid tumors), ascites, using withdrawal, lavage, wash, surgical dissection, etc. Cells can be obtained from fixed or unfixed, fresh or frozen, whole or disintegrated samples. Disintegration of tissues can occur either mechanically or enzymatically using known techniques.

[0104] Imaging The disclosed methods include imaging amplicons embedded in one or more hydrogels using any of several different types of microscopy, such as confocal microscopy, two-photon microscopy, bright-field microscopy, intact tissue expansion microscopy, and / or CLARITY™ Optimized Light Sheet Microscopy (COLM).

[0105] Bright-field microscopy is the simplest of all optical microscopy techniques. Sample illumination is via transmitted white light, i.e. illuminated from below and observed from above. Limitations include the low contrast of most biological samples and the poor apparent resolution due to blurring of out-of-focus material. The simplicity of the technique and the minimal sample preparation required are key advantages.

[0106] In oblique illumination microscopy, the specimen is illuminated from the side. This gives the image a three-dimensional appearance and can highlight features that would not otherwise be visible. A more recent technique based on this method is Hoffman's Modulation Contrast, a system found on inverted microscopes for use with cell cultures. Oblique illumination suffers from the same limitations as bright-field microscopy (low contrast in many biological specimens due to blurring of out-of-focus material, and low apparent resolution), but it can highlight structures that would not otherwise be visible.

[0107] Dark field microscopy is a technique for improving the contrast of unstained, transparent specimens. Dark field illumination uses a carefully aligned light source to minimize the amount of directly transmitted (unscattered) light entering the image plane, collecting only the light scattered by the specimen. Dark field can dramatically improve image contrast (especially of transparent objects) with little need for instrument setup or specimen preparation. However, the technique suffers from low light intensity in the final image for many biological specimens and continues to suffer from poor apparent resolution.

[0108] Phase contrast is an optical microscopy illumination technique in which the phase shift of light passing through a transparent specimen is converted into a change in brightness in the image. That is, phase contrast shows differences in the refractive index as differences in contrast. The phase shift itself is invisible to the human eye, but becomes visible as the brightness changes.

[0109] In differential interference contrast (DIC) microscopy, differences in optical density are shown as differences in relief. The system consists of a special prism (Nomarski prism, Wollaston prism) in a condenser, which splits the light into ordinary and extraordinary beams. The spatial difference between the two beams is minimal (less than the maximum resolution of the objective). After passing through the specimen, the beams are recombined by a similar prism in the objective. In homogeneous specimens, there is no difference between the two beams and no contrast is produced. However, near refractive boundaries (e.g., the nucleus in the cytoplasm), the difference between the ordinary and extraordinary beams causes relaxation in the image. Differential interference contrast requires a polarized light source to work and two polarizing filters must be installed in the light path, one below the condenser (polarizer) and one above the objective (analyzer).

[0110] Another microscopy technique that uses interference is interference reflectance microscopy (also known as reflective interference contrast, or RIC). It is used to study the adhesion of cells to glass surfaces, using polarized light of a narrow range of wavelengths that is reflected whenever there is an interface between two materials with different refractive indices. When cells bind to the glass surface, the light reflected from the glass interferes with the light reflected from the bound cells. If there are no cells bound to the glass, there is no interference.

[0111] A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study the properties of organic or inorganic matter. In fluorescence microscopy, a sample is illuminated with light of a wavelength that excites fluorescence in the sample. The fluorescent light, which is usually at a longer wavelength than the illumination, is then imaged through a microscope objective. Two filters can be used in this technique: an illumination (or excitation) filter, which ensures that the illumination is close to monochromatic and of the correct wavelength, and a second emission (or barrier) filter, which ensures that none of the excitation light source reaches the detector. Alternatively, both of these functions can be accomplished by a single dichroic filter. "Fluorescence microscope" refers to any microscope that uses fluorescence to produce images, whether it is a simpler setup like a fluorescent microscope, or a more complex design like a confocal microscope that uses optical sectioning to obtain better resolution of the fluorescent image.

[0112] Confocal microscopes use point illumination and a pinhole in an optically bonded plane in front of the detector to eliminate out-of-focus signals. Because only light produced by fluorescence very close to the focal plane can be detected, the optical resolution of the image, especially in the sample depth direction, is much better than that of wide-field microscopes. However, this increased resolution comes at the expense of a reduction in signal intensity, as much of the light from the sample fluorescence is blocked at the pinhole, so long exposure times are often required. Because only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning over a regular raster (i.e., a rectangular pattern of parallel scan lines) in the specimen. The achievable thickness of the focal plane is defined primarily by the wavelength of the light used divided by the numerical aperture of the objective lens, but also by the optical properties of the specimen. The thin optical sections possible make these types of microscopes particularly excellent for 3D imaging and surface profiling of samples. COLM offers an alternative microscopy method for fast 3D imaging of large clarified samples. COLM allows the investigation of large immunostained tissues, improving the speed of acquisition and resulting in a higher quality of the data generated.

[0113] In single plane illumination microscopy (SPIM), also known as light sheet microscopy, only fluorophores in the focal plane of the detection objective are illuminated. The light sheet is a beam collimated in one direction and focused in the other. The method also offers unique optical sectioning, since no fluorophores are excited outside the focal plane of the detector. Furthermore, compared to traditional microscopy, light sheet methods exhibit reduced photobleaching and reduced phototoxicity, often allowing many more scans per specimen. By rotating the specimen, the technique can image virtually any plane, with multiple views obtained from different angles. However, for all angles, only a relatively shallow section of the specimen is imaged with high resolution, while deeper regions appear increasingly blurred.

[0114] Super-resolution microscopy is a form of optical microscopy. Due to the diffraction of light, the resolution of conventional optical microscopy is limited, as stated by Ernst Abbe in 1873. A good approximation of the achievable resolution is the FWHM (full width at half maximum) of the point spread function, and precise wide-field microscopes with high apertures and visible light typically reach a resolution of about 250 nm. Super-resolution techniques allow images to be captured with a resolution higher than the diffraction limit. These are divided into two broad categories: "true" super-resolution techniques, which capture the information contained in the evanescent waves, and "functional" super-resolution techniques, which use experimental techniques and known limitations of the material being imaged to reconstruct a super-resolution image.

[0115] Laser microscopy uses laser illumination sources in various forms of microscopy. For example, laser microscopy focused on biological applications uses ultrashort pulsed lasers, or femtosecond lasers, in several techniques including nonlinear microscopy, saturation microscopy, and multiphoton fluorescence microscopy such as two-photon excitation microscopy (a fluorescence imaging method that allows imaging of living tissues at very high depths, e.g., up to 1 millimeter).

[0116] Electron microscopy (EM) uses a beam of electrons to illuminate a specimen and produce a magnified image. Electron microscopes have a wavelength about 100,000 times shorter than visible light (photons), and therefore have higher resolution than light-powered optical microscopes. They can achieve resolutions better than 50 pm and magnifications up to about 10,000,000 times, whereas ordinary unfocused light microscopes are limited by diffraction to a resolution of about 200 nm and useful magnifications of less than 2000 times. Electron microscopes use electrostatic and electromagnetic "lenses" to control and focus the electron beam to form an image. These lenses are similar to, but different from, the glass lenses of a light microscope, which form a magnified image by focusing light on or through the specimen. Electron microscopes are used to observe a wide range of biological and inorganic specimens, including microorganisms, cells, large molecules, biopsy samples, metals, and crystals. Industrially, electron microscopes are often used for quality control and failure analysis. Examples of electron microscopy include transmission electron microscopy (TEM), scanning electron microscopy (SEM), reflection electron microscopy (REM), scanning transmission electron microscopy (STEM) and low voltage electron microscopy (LVEM).

[0117] Scanning probe microscopy (SPM) is a branch of microscopy that uses a physical probe to scan a specimen to form an image of a surface. The image of the surface is obtained by mechanically moving the probe line by line in a raster scan of the specimen and recording the interaction of the probe with the surface as a function of position. Examples of SPM include atomic force microscopy (ATM), ballistic electron emission microscopy (BEEM), chemical force microscopy (CFM), conductive atomic force microscopy (C-AFM), electrochemical scanning tunneling microscopy (ECSTM), electrostatic force microscopy (EFM), fluid force microscopy (FluidFM), force modulation microscopy (FMM), function-oriented scanning probe microscopy (FOSP), Kelvin probe force microscopy (KPFM), magnetic force microscopy (MFM), magnetic resonance force microscopy (MRFM), near-field scanning optical microscopy (NSOM) (or SNOM, scanning near-field optical microscope, SNOM, piezoresponse force microscopy (PFM), PSTM, photon scanning tunneling microscopy (PSTM), PTMS, photothermal microscopy. Spectroscopy / Microscopy (PTMS), SCM, Scanning Capacitance Microscopy (SCM), SECM, Scanning Electrochemical Microscopy (SECM), SGM, Scanning Gate Microscopy (SGM), SHPM, Scanning Hall Probe Microscopy (SHPM), SICM, Scanning Ion Conductance Microscopy (SICM), SPSM Spin Polarized Scanning Tunneling Microscopy (SPSM), SSRM, Scanning Diffusion Resistance Microscopy (SSRM), SThM, Scanning Thermal Microscopy (SThM), STM, Scanning Tunneling Microscopy (STM), STP, Scanning Tunneling Potentiometry (STP), SVM, Scanning Voltage Microscopy (SVM), and Synchrotron X-ray Scanning Tunneling Microscopy (SXSTM).

[0118] Intact tissue expansion microscopy (exM) allows imaging of thick preserved specimens with a lateral resolution of about 70 nm. With ExM, the optical diffraction limit is circumvented by physically expanding the biological specimen before imaging, thus bringing sub-diffraction-limited structures into the size range visible by conventional diffraction-limited microscopes. ExM can image biological specimens at the voxel rate of diffraction-limited microscopes, but at the voxel size of super-resolution microscopes. Expanded samples are transparent and refractive index-matched to water, since the expanded material is >99% water. Expansion microscopy techniques are known in the art, for example, as disclosed in Gao et al., Q&A: Expansion Microscopy, BMC Biol. 2017;15:50.

[0119] Screening Methods The methods disclosed herein also provide a method of screening a candidate agent to determine whether the candidate agent modulates gene expression of a nucleic acid in a cell in an intact tissue, the method including performing the steps of STARmap2 disclosed herein to determine a gene sequence of a target nucleic acid in a cell in the intact tissue, and detecting a level of gene expression of the target nucleic acid, where a change in the level of expression of the target nucleic acid in the presence of the candidate agent relative to the level of expression of the target nucleic acid in the absence of the candidate agent indicates that the candidate agent modulates gene expression of the nucleic acid in a cell in the intact tissue.

[0120] In some embodiments, the detecting comprises performing flow cytometry, sequencing, probe binding and electrochemical detection, pH change, catalysis induced by enzymes bound to DNA tags, quantum entanglement, Raman spectroscopy, terahertz wave technology, and / or scanning electron microscopy. In certain embodiments, the flow cytometry is mass cytometry or fluorescence activated flow cytometry. In some other embodiments, the detecting comprises performing microscopy, scanning mass spectrometry, or other imaging techniques described herein. In such embodiments, the detecting comprises determining a signal, e.g., a fluorescent signal.

[0121] As used interchangeably herein, the terms "test agent," "candidate agent," and grammatical equivalents refer to any molecule (e.g., a protein (which includes herein proteins, polypeptides, and peptides), a small molecule (i.e., between 5-1000 Da, 100-750 Da, 200-500 Da, or less than 500 Da in size), or an organic or inorganic molecule, polysaccharide, polynucleotide, etc.) that is tested for activity in the subject assay.

[0122] A variety of different candidate agents can be screened by the above methods. Candidate agents include numerous chemical classes, such as small organic compounds having molecular weights of more than 50 Daltons (e.g., at least about 50 Da, at least about 100 Da, at least about 150 Da, at least about 200 Da, at least about 250 Da, or at least about 500 Da), and less than about 20,000 Daltons, less than about 10,000 Daltons, less than about 5,000 Daltons, or less than about 2,500 Daltons. For example, in some embodiments, suitable candidate drugs are organic compounds having a molecular weight in the range of about 500 Da to about 20,000 Da, e.g., about 500 Da to about 1000 Da, about 1000 Da to about 2000 Da, about 2000 Da to about 2500 Da, about 2500 Da to about 5000 Da, about 5000 Da to about 10,000 Da, or about 10,000 Da to about 20,000 Da.

[0123] Candidate agents may contain functional groups necessary for structural interaction with proteins, e.g., hydrogen bonding, and may include at least an amine, carbonyl, hydroxyl, or carboxyl group, or at least two of the functional chemical groups. Candidate agents may include cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

[0124] Candidate agents can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. Numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including, for example, expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds can be readily modified through conventional chemical, physical and biochemical means and used to generate combinatorial libraries. Known pharmacological agents can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidation, etc., to generate structural analogs. Additionally, screening can be directed to known pharmacologically active compounds and their chemical analogs, or to novel agents with unknown properties, such as those generated through rational drug design.

[0125] In one embodiment, the candidate modulator is a synthetic compound.Any number of means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides.See, for example, WO94 / 24314, which is expressly incorporated herein by reference and discusses methods for generating novel compounds, including random chemical and enzymatic methods.

[0126] In another embodiment, candidate agents are provided as libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts that are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents can be subjected to directed or random chemical modification, including enzymatic modification, to produce structural analogs.

[0127] In one embodiment, candidate agents include proteins (including antibodies, antibody fragments (i.e., fragments containing antigen binding regions, single chain antibodies, etc.), nucleic acids, and chemical moieties). In one embodiment, candidate agents are naturally occurring proteins or fragments of naturally occurring proteins. Thus, for example, cellular extracts containing the proteins, or random or directed digests of proteinaceous cellular extracts, may be tested. In this manner, libraries of prokaryotic and eukaryotic proteins may be generated for screening. Other embodiments include libraries of bacterial, fungal, viral, and mammalian proteins (e.g., human proteins).

[0128] In one embodiment, the candidate agent is an organic moiety. In this embodiment, the candidate agent is synthesized from a series of substrates that can be chemically modified, generally as described in WO94 / 24314. "Chemical modification" as used herein includes conventional chemical and enzymatic reactions. These substrates generally include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes, and heteroalkyls), aryl groups (including arenes and heteroaryls), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepines, beta-lactams, tetracyline, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecodysone, and the like), alkaloids (including ergots, vincas, curares, pyrrolizudines, and mitomycins), organometallic compounds, heteroatom-bearing compounds, amino acids, and nucleosides. Chemical (including enzymatic) reactions can be performed on moieties to form new substrates or candidate drugs, which can then be tested using the present invention.

[0129] Devices and Systems Also included are devices for carrying out aspects of the methods. The devices may include, for example, imaging chambers, electrophoresis apparatus, flow chambers, microscopes, needles, tubes, pumps.

[0130] The present disclosure also provides systems for carrying out the subject methods. The systems may include, for example, a power source, a refrigeration unit, a waste, a heating unit, a pump, etc. The systems may also include any of the reagents described herein, for example, imaging buffer, washing buffer, stripping buffer, Nissl and DAPI solutions. Systems according to certain embodiments may also include a microscope and / or associated imaging equipment, for example, a camera component, a digital imaging component and / or an image capture device, a computer processor configured to collect images according to one or more user inputs, etc.

[0131] As discussed above, the system described herein comprises a fluidic device having an imaging chamber and a pump, and a processor unit configured to execute the method for in situ gene sequencing of target nucleic acids in cells in intact tissues described herein. In some embodiments, the system allows for automation of the process described herein, STARmap2, including, but not limited to, repeated rounds of hybridization of probes with DNA embedded in a gel, ligation of fluorescently labeled oligonucleotides on these probes, washing away excess probes, imaging, and peeling away probes for the next round of sequencing. In some embodiments, the system may allow for continuous operation. In some embodiments, the system includes an imaging chamber for flowing sequencing chemicals involved in in situ DNA sequencing to the sample. In some embodiments, a system of fluids and pumps controls the delivery of sequencing chemicals to the sample.

[0132] Buffers may be added / removed / recirculated / exchanged through the use of one or more ports and, optionally, tubing, pumps, valves, or any other suitable fluid handling and / or fluid manipulation equipment, e.g., tubing that is removably or permanently attached to one or more components of the device. For example, a first tube having a first and second end may be attached to a first port, and a second tube having a first and second end may be attached to a second port, with the first end of the first tube attached to the first port and the second end of the first tube operably connected to a container, e.g., a cooling unit, a heating unit, a filtration unit, a waste container, etc., and the first end of the second tube attached to the second port and the second end of the second tube operably connected to a container, e.g., a cooling unit, an ice beaker, a filtration unit, a waste container, etc.

[0133] In some embodiments, the system includes a non-transitory computer readable storage medium having instructions that, when executed by the processor unit, cause the processor unit to control the delivery of chemicals and synchronize this process with the microscope. In some embodiments, the non-transitory computer readable storage medium includes instructions that, when executed by the processor unit, cause the processor unit to measure an optical signal.

[0134] usefulness The devices, methods, and systems herein find several uses in the art, such as biomedical research and / or clinical diagnostics. For example, in biomedical research, applications include, but are not limited to, spatially resolved gene expression analysis for basic biology or drug screening. In clinical diagnostics, applications include, but are not limited to, detection of genetic markers such as disease, immune response, bacterial or viral DNA / RNA in patient samples. Examples of advantages of the methods described herein include efficiency, taking only 3 or 4 days to obtain final data from a raw sample, providing much faster speeds than existing microarray or sequencing technologies, being highly multiplexed (up to 1000 genes), single cell and single molecule sensitivity, preserved tissue morphology, and / or high signal-to-noise ratio with low error rate.

[0135] In certain aspects, STARmap2 can be applied to study molecularly defined cell types and activity-regulated gene expression in mouse visual cortex, and can be extended to larger 3D tissue blocks to visualize short- and long-range spatial organization of cortical neurons at volumetric scales not previously accessible. In some embodiments, the methods disclosed herein can be adapted to image DNA-conjugated antibodies for highly multiplexed protein detection.

[0136] The devices, methods, and systems of the present invention can also be generalized to study several heterogeneous cell populations in various tissues. Without being bound by any scientific theory, the brain poses special challenges that are highly suitable for STARmap2 analysis. For example, polymorphic activity-regulated gene (ARG) expression observed across different cell types is likely to depend on both intrinsic cell biological properties (e.g., signaling pathway component expression) and external properties such as neural circuit anatomy that sends external sensory information to different cells (here, the visual cortex). In such cases, in situ transcriptomics, as exemplified by STARmap2, can effectively link imaging-based molecular information with anatomical and activity information, thus elucidating brain function and dysfunction.

[0137] The devices, methods, and systems disclosed herein allow cellular components, such as lipids that normally provide structural support but impede the visualization of subcellular proteins and molecules, to be removed while maintaining the three-dimensional structure of cells and tissues because the sample is crosslinked to a hydrogel that physically supports the tissue ultrastructure. This removal essentially renders the interior of the biological specimen transparent to light and / or macromolecules, allowing the interior of the specimen, such as cells and subcellular structures, to be visualized microscopically without time-consuming destructive cutting of the tissue. This procedure is faster than procedures commonly used in the art, since clearing and permeabilization, which are typically performed in separate steps, can be combined in a single step that removes cellular components. In addition, for comprehensive analysis, the specimen can be repeatedly stained, unstained, and restained with other reagents. Further functionalization with polymerizable acrylamide moieties allows amplicons to be covalently fixed within the polyacrylamide network at multiple sites.

[0138] In one example, the subject devices, methods, and systems may be used to evaluate, diagnose, or monitor a disease. As used herein, "diagnosis" generally includes predicting a subject's susceptibility to a disease or disorder, determining whether a subject is currently affected by a disease or disorder, predicting a subject affected by a disease or disorder (e.g., identifying the cancer state, stage of cancer, the likelihood that a patient will die from cancer), predicting a subject's responsiveness to treatment of a disease or disorder (e.g., positive response, negative response, e.g., no response at all to allogeneic hematopoietic stem cell transplantation, chemotherapy, radiation therapy, antibody therapy, small molecule compound therapy), and using a therapy (e.g., monitoring a subject's condition to provide information regarding the efficacy or effectiveness of a therapy). For example, a biopsy may be prepared from the cancer tissue and microscopically analyzed to determine the type of cancer, the extent to which the cancer has developed, and whether the cancer will respond to a therapeutic intervention.

[0139] The subject devices, methods, and systems also provide useful techniques for screening candidate therapeutic agents for their effects on tissues or diseases. For example, a subject, e.g., a mouse, rat, dog, primate, human, etc., can be contacted with a candidate agent, its organ or biopsy can be prepared by the subject method, and the prepared specimen can be analyzed microscopically for one or more cellular or tissue parameters. A parameter is a quantifiable component of a cell or tissue, particularly one that can be accurately measured, preferably in a high-throughput system. A parameter can be any cellular component or cellular product, including cell surface determinants, receptors, proteins or their conformational or post-translational modifications, lipids, carbohydrates, organic or inorganic molecules, nucleic acids, e.g., mRNA, DNA, etc., or moieties derived from such cellular components or combinations thereof. Most parameters provide a quantitative readout, although in some cases, semi-quantitative or qualitative results are acceptable. The readout may include a single determined value, or may include the mean, median, or variance, etc. Characteristically, a range of parameter readouts is obtained for each parameter from multiplexes of the same assay. Variability is expected, and the range of values ​​for each set of test parameters is obtained using standard statistical methods with common statistical methods used to provide a single value. Thus, for example, one such method may include detecting cell viability, tissue vascularization, the presence of immune cell infiltration, effects that alter disease progression, and the like. In some embodiments, the screen includes comparing the analyzed parameters to those from a control, or reference sample, such as a specimen similarly prepared from a subject not in contact with the candidate agent. Candidate agents of interest for screening include known and unknown compounds encompassing numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, gene sequences, and the like. Candidate agents of interest for screening also include nucleic acids, such as nucleic acids encoding siRNAs, shRNAs, antisense molecules, or miRNAs, or nucleic acids encoding polypeptides. An important aspect of the present invention is the evaluation of candidate drugs, including toxicity testing, and the like.Evaluation of tissue samples using the subject methods can include, for example, genetic, transcriptional, genomic, proteomic, and / or metabolomic analyses.

[0140] The subject devices, methods, and systems may also be used to visualize the distribution of genetically encoded markers in whole tissues at subcellular resolution, such as chromosomal abnormalities (inversions, duplications, translocations, etc.), loss of genetic heterozygosity, the presence of genetic alleles indicative of a predisposition to disease or good health, likely responsiveness to therapy, ancestry, etc. Such detection may be used, for example, in the diagnosis and monitoring of diseases as described above, personalized medicine, and paternity studies.

[0141] Databases of analytical information may be accumulated. These databases may include results obtained from known cell types, references from analysis of cells treated under specific conditions, etc. A data matrix may be generated, where each point in the data matrix corresponds to a readout from a cell, and the data for each cell may include readouts from multiple labels. The readouts may be the mean, median, or variance, or other statistically or mathematically derived values ​​associated with the measurements. The output readouts may be further refined by direct comparison with the corresponding reference readouts. The absolute values ​​obtained for each output under identical conditions indicate the inherent variability of the biological system, and also reflect the variability of individual cells and the inherent variability between individuals.

[0142] Examples of Non-Limiting Aspects of the Disclosure The aspects (including embodiments) of the present subject matter described above may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the above description, certain non-limiting aspects of the present disclosure, numbered 1-71, are provided below. As will be apparent to one of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding aspects or any of the aspects following the individually numbered aspect. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below.

[0143] 1. A method for in situ gene sequencing of a target nucleic acid in cells in an intact tissue, comprising: (a) contacting a fixed, permeabilized, intact tissue with at least one pair of oligonucleotide primers under conditions that permit specific hybridization; the pair of primers comprises a first oligonucleotide and a second oligonucleotide; each of the first oligonucleotide and the second oligonucleotide comprises a first region of complementarity, a second region of complementarity, and a third region of complementarity, the second oligonucleotide further comprising a barcode sequence; contacting a first oligonucleotide, wherein a first complementary region of the first oligonucleotide is complementary to a first portion of the target nucleic acid, a second complementary region of the first oligonucleotide is complementary to a first complementary region of the second oligonucleotide, a third complementary region of the first oligonucleotide is complementary to a third complementary region of the second oligonucleotide, and a second complementary region of the second oligonucleotide is complementary to a second portion of the target nucleic acid, the first portion of the target nucleic acid is adjacent to the second portion of the target nucleic acid, the second complementary region of the first oligonucleotide comprises a first portion of a unique matching sequence, the third complementary region of the first oligonucleotide comprises a second portion of a unique matching sequence, the first complementary region of the second oligonucleotide comprises a sequence that is complementary to the first portion of the unique matching sequence, and the third complementary region of the second oligonucleotide comprises a sequence that is complementary to the second portion of the unique matching sequence; (b) adding a ligase to ligate a second oligonucleotide to form a closed nucleic acid circle; (c) performing rolling circle amplification in the presence of the nucleic acid molecule, comprising using the second oligonucleotide as a template and the first oligonucleotide as a primer for a polymerase to form one or more amplicons; (d) embedding the one or more amplicons in the presence of hydrogel subunits to form one or more hydrogel-embedded amplicons; (e) contacting one or more hydrogel-embedded amplicons having barcode sequences with a set of sequencing primers under conditions that allow for ligation, the set of sequencing primers including a third oligonucleotide configured to decode bases and a fourth oligonucleotide configured to convert the decoded bases into a signal, and ligation occurs only when both the third oligonucleotide and the fourth oligonucleotide are complementary to adjacent sequences of the same amplicon; (f) repeating step (e) a number of times; and (g) imaging the amplicons embedded in the one or more hydrogels to determine in situ gene sequencing of the target nucleic acid in cells in the intact tissue; A method comprising: 2. The method of embodiment 1, further comprising contacting the fixed, permeabilized intact tissue with a gel adaptor oligonucleotide that binds to a first oligonucleotide, wherein the gel adaptor oligonucleotide comprises a nucleotide modification at the 5' or 3' end that links the gel adaptor oligonucleotide to a hydrogel during gelation. 3. The method of embodiment 2, wherein the nucleotide modification comprises an acrydite group. 4. The method of embodiment 2 or 3, wherein the first oligonucleotide further comprises a common binding site for a gel adaptor oligonucleotide. 5. The method of embodiment 4, wherein the common binding site for the gel adaptor oligonucleotide is adjacent to the first complementary region of the first oligonucleotide.

[0144] 6. The method of any one of aspects 1-5, further comprising contacting the fixed, permeabilized intact tissue with an oligonucleotide probe for detection and condensation of amplicons, wherein the oligonucleotide probe binds to a second oligonucleotide. 7. The method of embodiment 6, wherein the second oligonucleotide further comprises a common binding site for an oligonucleotide probe for detection and condensation of the amplicon. 8. The method of embodiment 7, wherein the common binding site for the oligonucleotide probes for detection and condensation of the amplicons is adjacent to a second region of complementarity or is adjacent to a sequence that is complementary to the second half of the unique matching sequence of the second oligonucleotide. 9. The method of any one of aspects 6-8, wherein the oligonucleotide probe comprises a unique sequence for detecting an amplicon of the intended probe target and two or more copies of a sequence complementary to a common sequence on the amplicon. 10. The method of any one of aspects 6 to 9, wherein the oligonucleotide probe further comprises a nucleotide modification at the 5' or 3' end such that the first oligonucleotide probe is linked to the hydrogel during gelation.

[0145] 11. The method of embodiment 10, wherein the nucleotide modification comprises an acrydite group. 12. Cells a first probe comprising a 5' amine modification or a 5' biotin modification, a common gel adapter complementary sequence that hybridizes to a gel adapter oligonucleotide, and a unique barcode sequence; and 12. The method of any one of aspects 1-11, further comprising barcoding the cells by contacting the cells with a second probe comprising a first sequence that is complementary to a first portion of the unique barcode sequence and a second sequence that is complementary to a second portion of the unique barcode sequence, wherein the first sequence and the second sequence are adjacent to a sequencing encoding sequence, and hybridization of the first probe and the second probe results in formation of a barcode complex comprising the first probe and the second probe. 13. The method of embodiment 12, wherein the second probe is a padlock probe. 14. The method of any one of aspects 1-13, wherein the first portion of the target nucleic acid and the second portion of the target nucleic acid have approximately the same melting temperature. 15. The method further comprises contacting the fixed and permeabilized intact tissue with an mRNA carrying oligonucleotide; The mRNA-carrying oligonucleotide A nucleotide modification at the 5' or 3' end, which allows the mRNA carrying oligonucleotide to be linked to the hydrogel during gelation; a poly-T tail that hybridizes to the poly-A tail of the mRNA, where hybridization of the poly-T tail of the mRNA retention oligonucleotide with the poly-A tail of the mRNA retains the mRNA in the hydrogel; and 15. The method of any one of embodiments 1 to 14, comprising a unique hybridization sequence.

[0146] 16. The method of embodiment 15, wherein the poly-T tail comprises reciprocal locked nucleic acid (LNA) thymine (T) bases. 17. The method of embodiment 15 or 16, further comprising contacting the fixed, permeabilized, intact tissue with a fluorescently labeled probe oligonucleotide that selectively binds to a unique hybridization sequence of the mRNA retention oligonucleotide. 18. The method according to any one of aspects 1 to 17, wherein sequencing is carried out using sequential or combinatorial encoding. 19. The method of any one of aspects 1-18, further comprising pre-incubating the tissue sample with polymerase for a sufficient time to allow uniform diffusion of the polymerase throughout the tissue prior to performing rolling circle amplification. 20. The method according to any one of aspects 1 to 19, wherein the imaging is carried out in the presence of an anti-fading buffer comprising an antioxidant.

[0147] 21. The method of claim 20, wherein the anti-fading buffer comprises Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and Trolox-quinone. 22. The method of any one of aspects 1 to 21, wherein the signal is a fluorescent signal. 23. The method of embodiment 22, further comprising removing the signal after imaging by contacting the hydrogel with formamide. 24. The method of embodiment 22, wherein the fourth oligonucleotide is covalently linked to the fluorophore by a disulfide bond. 25. The method of embodiment 24, further comprising contacting the hydrogel with a reducing agent after said imaging, wherein reduction of the disulfide bond results in cleavage of the fluorophore from the fourth oligonucleotide.

[0148] 26. The method of any one of aspects 1 to 25, wherein the set of primers is denatured by heating before contacting with the sample. 27. The method of any one of aspects 1 to 26, wherein the cell is present in a population of cells. 28. The method of embodiment 27, wherein the population of cells comprises multiple cell types. 29. The method of any one of aspects 1-28, wherein contacting the fixed, permeabilized intact tissue comprises hybridizing primers to the same target nucleic acid. 30. The method of any one of aspects 1 to 29, wherein the target nucleic acid is RNA or DNA.

[0149] 31. The method of embodiment 30, wherein the RNA is mRNA. 32. The method of any one of aspects 1-31, wherein the second oligonucleotide comprises a padlock probe. 33. The method of any one of aspects 1 to 32, wherein the first complementarity region of the first oligonucleotide has a length of 19 to 25 nucleotides. 34. The method of any one of aspects 1-33, wherein the second complementary region of the first oligonucleotide has a length of 6 nucleotides. 35. The method of any one of aspects 1-34, wherein the third complementary region of the first oligonucleotide has a length of 6 nucleotides.

[0150] 36. The method of any one of aspects 1-35, wherein the first complementary region of the second oligonucleotide has a length of 6 nucleotides. 37. The method of any one of aspects 1-36, wherein the second complementary region of the second oligonucleotide has a length of 19-25 nucleotides. 38. The method of any one of aspects 1 to 37, wherein the third complementary region of the second oligonucleotide has a length of 6 nucleotides. 39. The method of any one of aspects 1-38, wherein the first complementary region of the second oligonucleotide comprises the 5' end of the second oligonucleotide. 40. The method of any one of aspects 1-39, wherein the third region of complementarity of the second oligonucleotide comprises the 3' end of the second oligonucleotide.

[0151] 41. The method of any one of aspects 1-40, wherein the first complementary region of the second oligonucleotide is adjacent to the third complementary region of the second oligonucleotide. 42. The method of any one of aspects 1-41, wherein the barcode sequence of the second oligonucleotide provides barcode information for identification of the target nucleic acid. 43. The method of any one of aspects 1-42, wherein contacting the fixed, permeabilized intact tissue comprises hybridizing a plurality of oligonucleotide primers having specificity for different target nucleic acids. 44. The method of any one of aspects 1-43, wherein the second oligonucleotide is provided as a closed stranded nucleic acid circle and the step of adding a ligase is omitted. 45. Melting temperature (T m 45. The method of any one of embodiments 1 to 44, wherein the first and second coupling sites are selected to minimize ligation in solution.

[0152] 46. ​​The method of any one of aspects 1 to 45, wherein adding a ligase comprises adding a DNA ligase. 47. The method of any one of aspects 1 to 46, wherein the nucleic acid molecule comprises amine-modified nucleotides. 48. The method of embodiment 47, wherein the amine-modified nucleotide comprises an acrylate N-hydroxysuccinimide moiety modification. 49. The method of any one of aspects 1-48, wherein embedding comprises copolymerizing one or more amplicons with acrylamide. 50. The method of any one of aspects 1-49, wherein the embedding comprises clarifying the amplicons embedded in the one or more hydrogels, and the target nucleic acid is substantially retained in the amplicons embedded in the one or more hydrogels.

[0153] 51. The method of embodiment 50, wherein the clarifying comprises substantially removing a plurality of cellular components from the amplicons embedded in the one or more hydrogels. 52. The method of embodiment 50 or 51, wherein the clarifying comprises substantially removing lipids or proteins, or a combination thereof, from the amplicons embedded in the one or more hydrogels. 53. The method of any one of aspects 1-52, wherein contacting the amplicons embedded in one or more hydrogels comprises eliminating accumulation of errors as sequencing progresses. 54. The method of any one of aspects 1-53, wherein imaging comprises imaging the amplicons embedded in the one or more hydrogels using confocal microscopy, two-photon microscopy, bright-field microscopy, intact tissue expansion microscopy, and / or CLARITY™ Optimized Light Sheet Microscopy (COLM). 55. The method of any one of aspects 1-54, wherein the intact tissue is a thin section.

[0154] 56. The method of embodiment 55, wherein the intact tissue has a thickness of 5 to 20 μm. 57. The method of embodiment 55 or 56, wherein the contacting of the amplicons embedded in the one or more hydrogels occurs four or more times. 58. The method of any one of aspects 1-54, wherein the intact tissue is a thick section. 59. The method of embodiment 58, wherein the intact tissue has a thickness of 50 to 200 μm. 60. The method of embodiment 58 or 59, wherein the contacting of the amplicons embedded in one or more hydrogels occurs six or more times.

[0155] 61. The method of any one of aspects 1-60, wherein ligation of the third oligonucleotide and the fourth oligonucleotide is carried out in the presence of a polyethylene glycol polymer. 62. The method of embodiment 61, wherein the PEG polymer is PEG6000. 63. A method for screening a candidate agent to determine whether the candidate agent modulates gene expression of a nucleic acid in a cell in an intact tissue, comprising: Carrying out a method according to any one of aspects 1 to 62 to determine a genetic sequence of a target nucleic acid in a cell in an intact tissue; detecting a level of gene expression of the target nucleic acid, wherein a change in the level of expression of the target nucleic acid in the presence of the candidate agent relative to the level of expression of the target nucleic acid in the absence of the candidate agent indicates that the candidate agent modulates gene expression of the nucleic acid in cells in the intact tissue; A method comprising: 64. The method of embodiment 63, wherein detecting comprises performing flow cytometry, sequencing, probe binding and electrochemical detection, pH change, catalysis induced by an enzyme bound to a DNA tag, quantum entanglement, Raman spectroscopy, terahertz wave technology, and / or scanning electron microscopy. 65. The method according to embodiment 64, wherein the flow cytometry is mass cytometry or fluorescence activated flow cytometry.

[0156] 66. The method of any one of aspects 63-65, wherein detecting comprises performing microscopy, scanning mass spectrometry, or other imaging technique. 67. The method of any one of embodiments 63-66, wherein detecting comprises detecting a signal.

[0157] 68. The method of embodiment 67, wherein the signal is a fluorescent signal. 69. A system comprising: A fluidic device; A processor unit configured to carry out the method according to any one of aspects 1 to 68. A system comprising: 70. The system of embodiment 69, further comprising an imaging chamber. 71. The system of aspect 69 or 70, further comprising a pump.

[0158] experiment The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0159] All publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0160] The present invention has been described with respect to specific embodiments found or provided by the inventors to include preferred modes for carrying out the invention. Those skilled in the art will understand in light of this disclosure that numerous modifications and changes can be made in the specific embodiments exemplified without departing from the intended scope of the invention. For example, codon redundancy allows changes in the underlying DNA sequence to be made without affecting the protein sequence. Biological functional equivalence considerations allow changes in protein structure to be made without affecting biological action in type or amount. All such modifications are intended to be within the scope of the appended claims.

[0161] Example 1 STARmap2 next-generation volumetric in situ sequencing Introduction STARmap is a sequencing process in which target nucleic acids are selectively labeled with barcodes, which are amplified and attached to a hydrogel prior to subsequent multiple rounds of readout by ligation. The first publication of STARmap demonstrated the use of combinatorial sequencing on sample sizes as large as 0.0034 cubic millimeters, and sequential sequencing on sample sizes as large as 0.238 cubic millimeters. Collecting these data required significant human effort, with the sequencing readout stage alone requiring several days of continuous effort for a single sample. An entire adult mouse brain is approximately 400 cubic millimeters, or more than 110,000 times the volume of previously demonstrated combinatorial sequencing samples. As intact tissue sequencing approaches scale to increase capacity and throughput, it is critical to maximize their robustness (to prevent compound accumulation of errors) and enhance parallelism and throughput. This is especially true as these techniques move from the laboratory to clinical and industrial workflows.

[0162] STARmap2 next-generation volumetric in situ sequencing 1. Probe Design Ligation of each DNA moiety of the SNAIL complex is used as a scaffold to reduce background labeling by gene or probe pair-specific match signals 2. Adapter Oligos 3. Oligo retention and labeling 4. Chemical retention of targets Fixation and permeabilization conditions Other chemical modifications of the target Optional target retention on gel prior to hybridization and subsequent steps 5. Floating gel 6. Accelerated Ligation Using PEG 7.RCA conditions 8. Structuring and Self-Assembly of Amplicons Using Linker Oligos 9. Recombination of floating gel 10. Amplicon Binding 11. SCAL and SEDAL2 Sequencing for STARmap2 Reduced cycle times and simplified reagents for SCAL in STARmap2 Forward and reverse sequencing for highly efficient combined barcode reading in STARmap2

[0163] Technical description Probe Design Targeted probe-complementary sequence identification For a given species, a library of expressed genes was obtained by taking the shortest isoform for each gene from a library of all cDNA sequences for that species. A bowtie index was constructed from the shortest isoforms using Bowtie2. Candidate complementary sequences were extracted using OligoMiner's blockParse.py functionality in overlap mode with no temperature constraints, oligo lengths of 44-48 nucleotides (nt), and a minimum GC content of 30% and a maximum GC content of 70%. Candidate complementary sequences were then aligned to the bowtie index to check for uniqueness, and all non-unique sequences were removed from the set of candidate oligos. Finally, overlapping oligos were removed from the set of candidate genes, starting from the first position per gene.

[0164] Targeted probe-SNAIL construct For a given set of gene names, a library of SNAIL probes was designed, taking into account as input the target number of SNAIL pairs per gene (typically 4 or 16), as well as whether the encoding for sequencing should be continuous or combinatorial. For combinatorial encoding probe libraries, barcode sequences after the Hamming encoding scheme were pre-calculated for various code lengths and minimum distances, and based on the number of genes, an appropriate encoding set was selected (e.g., for a 200-gene set, a 7-length code with a minimum distance of 3 was used). Barcode sequences from this codebook were additionally filtered in the dibase encoding to not generate only a single color across all rounds. These filtered barcodes were assigned uniquely to each gene. Then, for both combinatorial and consecutive encoding, probe sequences were designed for each of the two SNAIL probe sequence parts, head and shell. Additionally, rather than using a common sequence across all probes in a probe set for the two adjacent complementary sites at the 3' end of the SNAIL head, as was done previously, unique matching sequences (SNAIL matching sequences), subject to GC constraints, were generated either per gene or per probe pair and used to complement each end of the SNAIL shell (the ends are hybridized together and then ligated). These SNAIL matching sequences have the effect of minimizing spurious probe-probe interactions that may arise due to either off-target hybridization, protein binding, or other probe aggregation or persistent close interactions (as the ligase rejects mismatched sequences), thus promoting specificity when a larger number of genes are targeted or higher probe concentrations are used. For a given probe pair, a unique sequence of the targeted gene was selected, reverse-complemented, and split into two parts, such that each part has an approximately equal melting temperature to the other part.A common 20 nt hybridization sequence (see gel adapter probes below) was followed by the first portion of the reverse-complemented target gene sequence, followed by a probe pair-specific or gene-specific SNAIL match sequence to form the SNAIL head. The second portion of the reverse complement of the target sequence was added after the reverse complement of the first half of the SNAIL match sequence, followed by sequencing the bases, and then adding the reverse complement of the second half of the SNAIL match sequence. For probe sets in which amplicon condensation and detection oligos were used (see thick section, library generation), additional sequences common to all SNAIL shells were added to the SNAIL shell sequence, either adjacent to the target-complementary sequence or adjacent to a unique SNAIL match sequence, so that the randomized N bases in the amplicon condensation and detection oligos tend to distinguish between hybridization targets (see thick section, library generation).

[0165] Gel Adapter Oligo An oligo complementary to the 5' hybridization sequence on the SNAIL head was designed with a functional linking modification at its 5' end, such as acrydite, to covalently link the SNAIL head sequence to the hydrogel during gelation (thus better retaining the resulting amplicon grown from the 3' end of this sequence) without requiring synthesis of the SNAIL head oligo with the 5' modification itself.

[0166] mRNA retention and labeling oligos The mRNA retention and labeling oligo was designed with a 5'-acrydite modification for retention in the hydrogel and a 14-mer polydt sequence containing reciprocal locked nucleic acid (LNA) T bases (for stable hybridization to the mRNA polyA tail), followed by a unique 18 nt hybridization sequence. This oligo can be used to retain the mRNA in the hydrogel during or before hybridization, and in some cases additionally allows gelation to be performed prior to hybridization. Importantly, the unique hybridization sequence on the oligo allows labeling of the total mRNA signal through hybridization with a complementary fluorophore-labeled oligo, even after the RNA has been degraded. This is important for segmentation based on cytoplasmic labeling in the post-sequencing data processing pipeline.

[0167] Cell barcoding A barcode per cell was designed with two components: first, a 5' splint sequence containing either a 5' amine modification (for immobilization or subsequent modification) or a 5' biotin modification (to facilitate polarized transport through the cell), a common gel adapter complementary sequence, and a unique 40 nt sequence, and second, a padlock probe containing at each end a 20 nt sequence complementary to each half of the unique 40 nt sequence of the first probe, flanked by sequencing encoding sequences (e.g., sequential encoding sequences for a particular round and base). The pair of components prehybridized together constitute a barcode that can be followed throughout the STARmap2 procedure of immobilization, hybridization (with the gel adapter oligo), polymerization into hydrogel, ligation, and amplification by RCA, allowing any endogenous signals to be detected and STARmap2 readout of the encoded sequence. For a methodological description of an exemplary use of these cellular barcodes for cell tagging and morphological reconstruction after patch clamp recordings in intact tissue volumes, see STARpatch below.

[0168] STARmap2 - Thin Section, Combined and Continuous Sample preparation Mice were deeply anesthetized using 5% isofluorane and decapitated. Brains were rapidly dissected from the skull, immersed in OCT on a chuck, flash frozen in a slurry of ethanol and dry ice, and stored at -80°C until use (at least 30 min before use). Prior to sectioning, brains were equilibrated to the cutting temperature in the cryostat for at least 15 min. Sections were cut to a thickness of 16 mm or less and transferred to wells of a glass-bottom plate that had been pretreated with Bind-Silane and poly-L-lysine according to the manufacturer's instructions. After tissue collection, sections were fixed in 4% paraformaldehyde (PFA) for 10 min to 1 h at room temperature (or up to 24 h at 4°C). Sections were then rinsed three times with ice-cold phosphate-buffered saline (PBS) and transferred to methanol that had been precooled to -20°C. Samples were stored at -80°C for at least 15 min and up to several months before use.

[0169] Sample Preparation - Binding to Slides Tissues previously collected on slides, with or without fixation, were then prepared as follows: If the tissue is FFPE and sectioned onto slides, standard paraffin removal with xylene, washing with ethanol, and rehydration of the sample are performed as typical for FFPE sample collection. If the sample is attached to a slide but not fixed, the sample is briefly fixed with 4% PFA at room temperature before proceeding. A hydrophobic barrier pen is used to draw a well around each sample on the glass slide, and subsequent sample preparation steps are performed in this well according to the library generation procedure outlined below. If the sample is over-fixed and can no longer be penetrated by enzymes, the following rehydration and hybridization and washing steps are performed first, followed by hydrogel embedding and clearing, which can then be separated from the glass slide using a razor or other instrument (because the proteinase has destroyed the binding of the tissue to the glass). Ligation, amplification, and re-embedding steps are then performed in a similar manner to the thick section protocol below.

[0170] Library Generation Sections were equilibrated to room temperature and rehydrated in PBS containing 0.1% Tween-20 and 0.1 U / ml Superase RNAse inhibitor (PBSTwR) for at least 20 minutes, followed by a second rinse in PBSTwR to remove any residual methanol. Samples were placed in hybridization buffer containing 2X saline sodium citrate (SSC) buffer, 10% formamide, 0.1% Tween-20, 0.1 mg / mL sheared salmon sperm DNA, probe sets (see targeted probes) suitable for combination or sequential sequencing at final concentrations of 1 nM to 100 nM per probe pair (probe aliquots heated to 90°C for 3 minutes and allowed to cool slowly to room temperature before being added to hybridization buffer), mRNA-labeled oligos at 3 uM, Superase RNAse inhibitor at a concentration of 0.2 U / ml, RNAse-free distilled and deionized water (HO), and sterile 10% dextran sulfate solution. In some cases, the dextran sulfate solution was omitted. At least 50 ml of hybridization buffer was added to the samples, typically 125 ml to 250 ml. Samples were incubated at 40°C for at least 12 hours and up to 48 hours in a humidified incubator (Shake-N-Bake hybridization oven) with agitation. After hybridization, sections were washed twice in PBSTwR at room temperature (RT) for 20 minutes each and agitated. A final stringency wash was performed in PBSTwR (supplemented with SSC to a 4x final concentration) for 20 minutes at 37°C and agitated. Sections were briefly rinsed in PBSTwR at RT. Sections were incubated in ligation mixture consisting of 0.2U / ml T4 DNA ligase, 1X T4 ligase buffer, 0.1mg / ml BSA, 0.2U / ml Superase RNAse inhibitor, and H2O. For some sections, ligation was performed using 7.5% PEG6000 to increase the ligation rate. In this case, the T4 DNA ligase buffer was formulated without DTT, which is not required for the reaction and precipitates in the PEG. Ligation was performed at room temperature and stirred for 2 hours.The sections were then washed twice with PBSTwR for 15 minutes each. The sections were then incubated in rolling circle amplification (RCA) buffer at 30°C. For combinatorial sequencing, the samples were incubated for 2 hours with agitation. For single amplicon-resolved sequential sequencing, the samples were also incubated for 2 hours with agitation. For sequential sequencing with total signal per cell, the samples were incubated for at least 2 hours and up to 24 hours. The RCA buffer consisted of 0.6U / ml Phi29 DNA polymerase, 1X Phi29 DNA polymerase buffer, 0.25mM dNTPs, 0.05mg / ml BSA, 40uM 5-(3-aminoallyl)-dUTP, 0.2U / ml Superase RNAse inhibitor, and H2O. The sections were then washed twice in PBSTw (without RNAse inhibitor) for 15 minutes each at room temperature without agitation. If necessary, samples were placed at 4°C until proceeding the next day. Samples were treated with 20 mM acrylic acid NHS ester (AA-NHS) buffer for 2 hours at room temperature without agitation. Samples were rinsed twice in PBSTw and the AA-NHS buffer was exchanged before being placed in prepolymerization buffer (PM1) consisting of 2X SSC, 4% acrylamide monomer, 0.2% bis-acrylamide monomer, and HO. Samples were incubated in PM1 for 30 minutes at room temperature to allow the monomer to penetrate the sections. After incubation, PM1 solution was removed from the sections and replaced with 10-20 ml ice-cold polymerization buffer (PM2) containing PM1 and 1:1000 v / v tetramethylethylenediamine (TEMED) and 1:1000 v / v ammonium persulfate (APS). PM2 was added to the sections on ice. A coverslip coated with Gel Slick solution according to the manufacturer's instructions was placed over the sections and pressed downward to remove air bubbles and minimize excess gel on the sections. Sections were allowed to polymerize at room temperature for 1-1.5 h.After the coverslip was removed with a pair of forceps and the sections were rinsed in PBSTw to remove unpolymerized monomers, the sections were incubated for at least 2 h and up to 24 h in digestion buffer containing 2X SSC, 2% SDS, 0.8 mg / ml proteinase K, 0.5% Triton X-100, and HO. Finally, the samples were washed three times in PBS for 5 min each to remove the digestion buffer and stained with DAPI at a concentration of 1:1000 for 10 min, then rinsed in PBS. In some cases, thin section samples were processed according to the following thick section protocol, except that the washing time was limited to 5-10 min per wash.

[0171] Preparation of STARmap2 – Thick Sections, Combined and Sequential Sample preparation Mice were perfused transcardially with ice-cold PBS followed by ice-cold 4% PFA. Brains were dissected and transferred to ice-cold 4% PFA for overnight fixation with agitation for up to 24 h. Brains were then rinsed in ice-cold PBS and then equilibrated at 4 °C for 30 min in ice-cold PBS. Brains were sectioned in fresh ice-cold PBS on ice on a vibratome to a thickness of 50-300 mm with a typical thickness of 150 mm. Sections containing the region of interest were transferred directly to ice-cold ethanol at 65% or more, typically 70% ethanol in HO. Sections were stored in ethanol solution at 4 °C for at least overnight and up to several months until use, but typically less than a month.

[0172] Library Generation Sections were equilibrated to room temperature and rehydrated in PBSTwR for at least 30 minutes, followed by a second rinse in PBSTwR to remove any residual ethanol. Samples were placed in hybridization buffer containing 2X saline sodium citrate (SSC) buffer, 10% formamide, 0.1 mg / mL sheared salmon sperm DNA, probe sets (see targeted probes) suitable for combination or sequential sequencing at final concentrations of 1 nM to 100 nM per probe pair (probe aliquots heated to 90°C for 3 minutes and allowed to cool slowly to room temperature before being added to hybridization buffer), mRNA labeling oligos at 3 mM, acrydite adaptor oligos (for gel retention of probe / RNA complexes) at equimolar concentrations of 1 / 2 to 2 times the total concentration of the probes, Superase RNAse inhibitor at a concentration of 0.2 U / ml, and RNAse-free distilled water, deionized water (HO), and 10% sterile dextran sulfate solution. At least 125 ml of hybridization buffer was added to the sample, typically 250 ml was used. Sections were incubated and agitated at 40°C for at least 12 hours, and typically 16-48 hours, in a humidified incubator (Shake-N-Bake hybridization oven). Sections were washed twice in PBSTwR supplemented with 2X SSC at 37°C for 30 minutes each, agitated. Sections were then washed in PBSTwR at room temperature for 30 minutes, agitated, and then rinsed in PBSTwR. The buffer was replaced with degassed PM1 containing 3% acrylamide and 0.15% bis-acrylamide, and sections were placed on ice for at least 1 hour to allow the PM1 monomer to penetrate the sections. PM1 was then removed and replaced with 40 ml PM2 (prepared on ice) while the sample remained on ice, and coverslipped with a Gel-Slick-coated coverslip. The coverslip was pressed downwards to remove any air bubbles and ensure uniform gelling with minimal gel on the sections, and the sections were left at room temperature for 1.5 hours to gel.In some cases, this gelation step was performed with sections in wells where the coverslips had been pretreated with bind silane according to the manufacturer's instructions so that gelation would attach the samples to the well plate glass, and the subsequent library preparation steps were performed with the samples immobilized on the glass. In other cases, the gelation step was performed without treated glass, in which case the rest of the sample preparation steps were performed with floating sections, which allowed better reagent diffusion into the sample (from both sides of the resulting gel), better uniformity of the resulting signal, and more efficient enzyme action due to increased diffusion. After gelation, the coverslip was removed with forceps and the sections were washed with PBSTwR before being transferred to digestion buffer as above and clarified by incubation at 37 °C for 16-24 h with agitation. In some cases, samples were digested for 48 h. Samples that did not appear optically clear were further clarified in fresh buffer. After digestion was complete, sections were washed either 3 times at room temperature with stirring in PBSTwR for 30 minutes or once at room temperature with stirring in PBS+2 mM PMSF solution for 30 minutes to inactivate proteinase K, then washed 2 times at room temperature with stirring in PBSTwR for 30 minutes. Sections were incubated in a ligation mixture consisting of 0.2U / ml T4 DNA ligase, 1X T4 ligase buffer, 0.1mg / ml BSA, 0.2U / ml Superase RNAse inhibitor, and H2O. For some sections, ligation was performed using 7.5% PEG6000 to increase the ligation rate. In this case, the T4 DNA ligase buffer was formulated without DTT. DTT is not required for the reaction and precipitates in the PEG. Ligation was performed at room temperature and stirred for 12-24 hours, typically 16 hours. The sections were then washed twice with PBSTwR for 30 min each with agitation at room temperature.Samples were preincubated for approximately 6 hours at 4° C. in RCA buffer consisting of 0.6 U / ml Phi29 DNA polymerase, 1× Phi29 DNA polymerase buffer, 0.25 mM dNTPs, 0.05 mg / ml BSA, 40 mM 5-(3-aminoallyl)-dUTP, 0.2 U / ml Superase RNAse inhibitor, and HO. In some cases, including thin section preparations and thick sections, the RCA buffer additionally contained amplicon condensation and detection oligos that may contain terminal modifications for binding to the gel such as 5′ primary amines or acrydites, and may contain unique sequences for amplicon detection, in addition to two or more copies of a sequence complementary to a common sequence on the amplicon, and one or more “N” bases placed next to each of the complementary sequences. A common sequence on the amplicon can be placed next to the sequence that first matches the probe target (e.g., endogenous mRNA) so that the amplicon condensation and detection oligo pool with randomized bases will tend to be sorted into oligos that are near or perfectly matched to the amplicon of a particular gene. This has the effect of pulling the strands together from a given amplicon for a particular gene (reducing the optical spread of the amplicon), making adjacent amplicons from different genes less likely to aggregate when tightly packed. Preincubation of the sample in the RCA mix (optionally without dNTPs) at 4 °C allows the polymerase to uniformly penetrate the sample at Z while minimizing degradation of the polymerase due to terminal degradation or instability of the extension oligos. After preincubation, the sample was transferred to 30 °C with rocking for 2 to 24 hours, typically 16 hours for sequential encoding and 3 hours for combinatorial encoding, because thick sections are optically more challenging and require greater amplification of signal compared to thin sections. If no degenerate oligos are used, a 3 hour incubation at 30° C. provides an optimal balance between signal intensity per amplicon and uniformity of amplicon shape.Amplicon condensation and detection oligos help ensure that increases in amplification time do not overwhelm downstream computational detection of amplicons due to amplicon size growth or irregularities in amplicon shape, especially for combinatorial sequencing in thick samples. After amplification, sections were washed twice with PBS for 20 minutes at room temperature without agitation. For samples where the previous step was performed on floating sections (in most cases), the sections were then attached to the glass bottom of the plate. To that end, in most cases the samples were transferred to poly-L-lysine coated glass bottom wells and oriented as desired. The buffer was then completely removed and the samples were allowed to dry somewhat flat on the glass (10 minutes at room temperature). The samples were then immersed in excess of amine-binding crosslinkers (typically with glutaraldehyde) such as PFA, glutaraldehyde, BSPEG9, or other Bis-NHS crosslinkers (with intermediate chains of varying length and composition) for 1 hour at room temperature, followed by quenching in Tris for 30 minutes and a final wash in PBS at room temperature three times for 20 minutes each, the second wash containing DAPI at 1:1000. In some cases, the samples were additionally stabilized on a coverslip via the addition of an elastomeric agent that rapidly polymerizes around the border of the sample. In some cases, the floating sections were incubated in AA-NHS buffer (as described in the thin section methods section) for 2 hours at room temperature, washed in PBS at room temperature for 20 minutes, and then incubated in degassed PM1 at room temperature for 30 minutes. Sections were then immersed in PM2 on ice for 5 min before being transferred to bindsilanized glass-bottom wells on ice, all PM2 solution removed, the specimen pressed flat against the well bottom, residual PM2 dried with a Kimwipe (to prevent over-gelation), and the specimen covered with a Gel-Slick coated coverslip, which was then allowed to polymerize for 1 h at room temperature, after which the coverslip was removed and the sections washed three times for 20 min in PBS at room temperature, the second wash containing DAPI at a concentration of 1:1000.For sections that did not float during the initial gelation but instead were attached to the glass bottom of the well, the samples were treated with a crosslinker as described above to better retain the amplicons, such as glutaraldehyde, PFA, BSPEG9, or other Bis-NHS crosslinkers, quenched with Tris, and then washed. Finally, in some cases, the entire thick section procedure was performed on samples that had previously undergone hydrogel formation and clarification, and targets such as splinted padlock probes and / or exogenous oligos (as barcodes or to label other cellular targets), endogenous RNA, or cellular components were retained in the hydrogel (e.g., the CLARITY procedure). Linking of these probes or targets to the gel was performed as described above for hydrogel binding, or by other chemical crosslinkers, specific antibodies, or oligos combined with hydrogel monomer moieties (e.g., by reacting an alkylating agent such as melphalan with acrylate NHS ester or acryloyl-X), modified to incorporate these substrates into the gel upon gel formation. The thick (or in some cases thin, where the final gelling step was omitted) section STARmap2 procedure was then performed starting from hybridization (in the case of non-splinted padlock probes) or from the ligation step, with the initial gelling and digestion steps removed.

[0173] Sequencing cycles - SCAL and SEDAL2 A sequencing cycle for SCAL or SEDAL2 optionally begins with a short sample wash before proceeding to the first signal addition. For SCAL sequencing, depending on whether sequential or combinatorial encoding is used for a particular round, the corresponding set of OR and fluor oligos or RO and fluor oligos and their round-specific competitors are added to the sample in a ligase mix consisting of BSA (1:100), T4 ligase (1:20), 10xT4 ligase buffer (as above, but without DTT), PEG6000 (7.5%), DAPI (1:1000), and HO. For combinatorial encoding, a RO oligo for a given position x is added, plus a dibase-encoding fluorophore set of 16 oligos, plus a competitor oligo for the previous position that was labeled (unless it is the first round of labeling, in which case the competitor oligo is omitted). In sequential encoding, the OR oligos, 4-channel fluorophore mix, and round x-1 competitor oligos for a given round x are added unless it is the first round of labeling. The presence of PEG in the sequencing ligation mix substantially accelerates signal addition to the target. The target is labeled for 1-4 hours in the labeling stage. The sample is then washed in 1X SSC buffer to remove excess fluorophores and OR / RO oligos, after which the buffer is exchanged into imaging buffer consisting of anti-fading solution. The anti-fading solution is a two part solution that is stored separately to maintain activity at room temperature for longer periods. The first component of the imaging buffer contains Tris HCl (125 mM), SSC (1X), HO, Trolox-quinone (TQ) solution (approximately 500 mM quinone in stock, 100 mM TQ in imaging buffer), 2 mM Trolox, glucose oxidase (0.66 mg / ml), and catalase (0.8 mg / ml). The second component of the imaging buffer is composed of 1.11 M glucose in 1X SSC. After incubation of the sample in the imaging buffer, the sample is imaged and briefly rinsed before proceeding to the next sequencing cycle.

[0174] For SEDAL2, the same oligo / ligation mixture is used as described above during the signal addition step, except that the competitor oligo is omitted and the OR / RO oligo does not contain a competitor-specific complementary sequence in the oligo. After sample addition, washing, imaging buffer addition, and imaging as described above, SEDAL2 includes a separate step for signal removal, where the signal is either stripped away with a formamide-containing stripping solution (e.g., 80% formamide, 0.1% Triton-X, 2X SSC, and H2O) or, if a thiol-linked dye is used to sequentially encode fluorescent oligos, with a cleavage solution containing 2X SSC, 50 mM TCEP, and H2O. The sample is then washed in either 1X SSC in H2O or PBSTw as described above before proceeding to the next round of signal addition.

[0175] Data Transfer Because a sequencing run can generate large amounts of data, a separate system thread runs during sequencing that periodically checks for completed data files (images, metadata, configuration) and streams them to either a large storage buffer disk or cloud computing storage where the data is further processed.

Claims

1. A method for identifying a nucleic acid, comprising: (a) contacting a cell containing said nucleic acid with a first pair of oligonucleotides under conditions sufficient to hybridize to said nucleic acid; The first pair of oligonucleotides comprises: i. a first oligonucleotide comprising a first complementary region, a second complementary region, and a third complementary region; ii. a second oligonucleotide comprising a fourth region of complementarity, a fifth region of complementarity, and a sixth region of complementarity, the second oligonucleotide comprising a first end and a second end; Contains contacting said first region of complementarity with a first portion of said nucleic acid, said second region of complementarity with said fourth region of complementarity, said third region of complementarity with said sixth region of complementarity, said fifth region of complementarity with a second portion of said nucleic acid, said first region of complementarity adjacent to said fifth region of complementarity, and said second oligonucleotide comprising a unique matching sequence that hybridizes to said first oligonucleotide; (b) ligating the first end and the second end to each other to generate a circular nucleic acid molecule; (c) amplifying the circular nucleic acid molecule to generate one or more amplicons; (d) imaging the one or more amplicons to identify the nucleic acid; A method comprising:

2. The method further comprising contacting the cell with a second pair of oligonucleotides, 2. The method of claim 1, wherein the second pair of oligonucleotides comprises a first oligonucleotide and a second oligonucleotide, the second oligonucleotide comprising a unique matching sequence, and the unique matching sequence of the second oligonucleotide of the second pair of oligonucleotides is different from the unique matching sequence of the second oligonucleotide of the first pair of oligonucleotides.

3. The method described in claim 2, wherein the first pair of oligonucleotides and the second pair of oligonucleotides bind to the same nucleic acid.

4. The method of claim 2, wherein the first pair of oligonucleotides and the second pair of oligonucleotides bind to different nucleic acids. (a) contacting the cell with two or more pairs of oligonucleotides, The cell comprises a plurality of nucleic acids, each pair of oligonucleotides of the two or more pairs of oligonucleotides recognizing one nucleic acid of the plurality of nucleic acids, and each pair of oligonucleotides of the two or more pairs of oligonucleotides recognizing one nucleic acid of the plurality of nucleic acids, i. a first oligonucleotide comprising a first complementary region, a second complementary region, and a third complementary region; ii. a second oligonucleotide comprising a fourth region of complementarity, a fifth region of complementarity, and a sixth region of complementarity, the second oligonucleotide comprising a first end and a second end; Contains contacting said first region of complementarity with a first portion of said nucleic acid, said second region of complementarity with said fourth region of complementarity, said third region of complementarity with said sixth region of complementarity, said fifth region of complementarity with a second portion of said nucleic acid, said first region of complementarity adjacent to said fifth region of complementarity, and said second oligonucleotide comprising a unique matching sequence that hybridizes to said first oligonucleotide; (b) ligating the first ends and second ends of two or more pairs of the oligonucleotides to each other to generate a plurality of circular nucleic acid molecules; (c) amplifying the plurality of circular nucleic acid molecules to generate a plurality of amplicons; (d) imaging said plurality of amplicons to identify said plurality of nucleic acids; The method of any one of claims 1 to 4, further comprising:

6. The method of claim 5, wherein (d) includes multiple rounds of imaging.

7. The method of claim 1, further comprising embedding the cells with one or more hydrogel subunits to form a hydrogel.

8. The method of claim 7, further comprising contacting the cell with a gel adaptor oligonucleotide, 8. The method of claim 7, wherein the gel adapter oligonucleotide comprises a 5' end and a 3' end, and the gel adapter oligonucleotide comprises a modification at the 5' end or the 3' end, which modification links the gel adapter oligonucleotide to the hydrogel during the embedding.

9. The method described in claim 8, wherein the first oligonucleotide further comprises a common binding site for the gel adapter oligonucleotide, and the common binding site for the gel adapter oligonucleotide is adjacent to the first complementary region of the first oligonucleotide.

10. The method of claim 1, further comprising contacting the cell with an mRNA-carrying oligonucleotide, The mRNA-retaining oligonucleotide comprises: (i) a nucleotide modification at the 5' or 3' end, wherein the mRNA-carrying oligonucleotide is linked to the hydrogel during the embedding; (ii) a poly-T tail that hybridizes to a poly-A tail of an mRNA, wherein hybridization of the poly-T tail of the mRNA-holding oligonucleotide with the poly-A tail of the mRNA holds the mRNA in the hydrogel; and (iii) a unique hybridization sequence; and The method of claim 8 , comprising:

11. The method of claim 10, wherein the poly-T tail comprises a reciprocal locked nucleic acid (LNA) thymine (T) base.

12. A method according to any one of claims 1 to 4, wherein the first complementarity region and the fifth complementarity region each have a melting temperature of 50 to 72°C.

13. The method of claim 1, wherein the imaging is carried out in the presence of an anti-fading buffer containing an antioxidant.

14. The method of any one of claims 1 to 4, wherein the one or more amplicons comprise a barcode.

15. The method of claim 1, further comprising contacting the one or more amplicons with a set of sequencing primers after (c) under conditions that permit ligation, 15. The method of claim 14, wherein the set of sequencing primers comprises a fifth oligonucleotide configured to decode a base and a sixth oligonucleotide configured to convert the decoded base into a signal, and the ligation occurs only when both the fifth oligonucleotide and the sixth oligonucleotide are complementary to adjacent sequences of one of the one or more amplicons.

16. The method of claim 15, wherein the signal is a fluorescent signal.

17. The method of claim 15, further comprising removing the signal after (d).

18. The method of claim 1, wherein the fourth complementarity region comprises a first portion of the unique matching sequence.

19. The method of claim 1, wherein the sixth complementarity region comprises a second portion of the unique matching sequence.

20. The method of claim 1, wherein the first complementarity region has a length of 19 to 25 nucleotides.

21. The method of claim 1, wherein the second complementary region has a length of 4 to 8 nucleotides.

22. A method described in any one of claims 1 to 4, wherein the third complementarity region has a length of 4 to 8 nucleotides.

23. The method of claim 1, wherein the fourth complementary region has a length of 4 to 8 nucleotides.

24. The method of claim 1, wherein the fifth complementarity region has a length of 19 to 25 nucleotides.

25. The method of claim 1, wherein the sixth complementarity region has a length of 4 to 8 nucleotides.

Images