Compositions and methods for genomic DNA and gene expression analysis in single cells

Abstract

Provided herein are compositions and methods for assessing the genomic landscape of fixed cells using photoactivated oligonucleotides that can be directed to the nucleus, mitochondria, or cytoplasm of fixed cells and, when activated, can be extended to replicate nuclear single-stranded DNA (i.e., open chromatin), open mitochondrial DNA, and / or cytoplasmic RNA in situ into barcoded complementary DNA. These methods also provide for gene-specific 3D chromatin structural niche analysis.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 898,824, filed on September 11, 2019, and U.S. Provisional Application No. 62 / 789,073, filed on January 7, 2019, the entire contents of each of which are incorporated herein by reference.

[0003] Statement Regarding Federally Funded Research

[0004] This invention was made with government support under Grant Nos. U01 MH098953, RM1 HG010023, and R01 MH110185 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] Reference to a sequence listing

[0006] This application contains a sequence listing, which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy was created on January 2, 2020, is named AGLTP0014WO_ST25.txt, and is 5 kilobytes in size. Background of the Invention 1. Technical Field

[0008] The present disclosure generally relates to methods and compositions for detecting open chromatin and / or RNA in single cells, as well as methods and compositions for detecting the 3D structure of chromatin at targeted locations. 2. Background technology

[0010] The spatial arrangement of genes, the structure of chromatin, and the accessibility of regulatory DNA elements control the expression of the nuclear architecture of the cell (Sherwood et al., 2014). The chromatin structure is then controlled by the epigenetic methylation of DNA, the modification of DNA binding proteins, and the dynamics of distal cis and trans chromosome regions. The organization of the genome is complex and dynamic. For example, through changes in chromatin folding, distal enhancers become very close to the promoters of regulated genes, and various groups estimate that there are millions of such potential enhancer interactions in the genome (Lai et al., 2015). This chromatin interaction of many genes has been mapped, and the mapping mainly uses cell populations, in which isolated nuclei are chemically cross-linked to maintain proximal promoter interactions. After cross-linking, antibodies against transcription factors or modified proteins (such as histones) are often used or simply by PCR for specific target sites to identify the target site (Simonis et al., 2006). The analysis of higher-order chromatin structure has become easier with the development of chromatin mapping procedures (chromosome-conformation-capture), including 3C, 4C, 5C, and HiC (de Wit and de Laat, 2012; Dekker et al., 2013). These procedures use restriction digestion of cross-linked chromatin, various PCR amplification strategies, and ligation of adapters to DNA, followed by sequencing of the products. Each of these processes becomes less selective due to the extensive manipulation of DNA. It is estimated that only 20% to 70% of cross-chromosome contacts can be captured using HiC cell populations. In addition, although HiC is better for chromosome topology analysis, it has a lower sensitivity, mainly due to the inefficiency of the ligation process.

[0011] A recent method for identifying open chromatin in single cells utilizes the assay for transposase accessible chromatin (ATACseq) (Buenrostro et al., 2013). This method uses the Tn5 transposase to mark accessible regions of the genome. When used on a single cell, single-cell ATACseq data is presented as merged data from multiple cells, because each transposon insertion only provides detection of a single allele region for each transposon, thereby prohibiting a second detection of the single allele region unless the data of multiple cells are aggregated. This program allows mapping of some regulatory sites, but it is reported that many sites previously identified by 3C / 4C are missed when targeting a large number of cells. In fact, only 9.4% of promoters are represented in ATAC. Further, Tn5 integration into the genome is not completely random, so some sequences will be missed. Finally, ATAC allows analysis on a genome-wide scale, with little ability to drive gene-specific analysis unless through random exploration. In order to assess this 3D structure at the cellular level, a method for assessing the open conformational state of the genome of a single cell is needed. Summary of the invention

[0012] Some embodiments provided herein relate to methods for assessing the multimodal genomic landscape of fixed cells using photoactivated oligonucleotides, which can be directed to the nucleus or cytoplasm of fixed cells and can be extended in situ when activated, thereby replicating nuclear single-stranded DNA (open chromatin) and / or cytoplasmic RNA into complementary DNA, such as barcoded complementary DNA. The separation, amplification and sequencing of these in situ transcribed cDNAs can provide information about how RNA is processed from the transcriptional potential of open genomic DNA into cytoplasmic steady-state RNA abundance in the context of how individual cells interact with their microenvironment. These methods also provide gene-specific 3D chromatin structural niche analysis, which can be used to identify spatially defined, biologically relevant and functional gene-specific enhancers.

[0013] In one embodiment, an oligonucleotide molecule is provided, which comprises an amplification segment, a hybridization segment, and a reversible termination nucleotide from 5' to 3'. In some aspects, the amplification segment is an RNA polymerase promoter. In some aspects, the amplification segment is a primer binding site. In some aspects, the amplification segment comprises about 7 to about 50 nucleotides. In some aspects, the hybridization segment comprises a random nucleotide sequence. In some aspects, the hybridization segment comprises a known nucleotide sequence. In some aspects, the known nucleotide sequence is complementary to the target genome or mitochondrial DNA sequence. In some aspects, the known nucleotide sequence is complementary to the target RNA sequence. In some aspects, the hybridization segment comprises a poly-T sequence. In some aspects, the hybridization segment comprises about 7 to about 30 nucleotides. In some aspects, the hybridization segment comprises about 15 nucleotides. In some aspects, the oligonucleotide also comprises an index barcode segment between the amplification segment and the hybridization segment. In some aspects, the oligonucleotide also comprises a spacer segment between the amplification segment and the index barcode segment. In some aspects, the reversible terminator nucleotide comprises a nitrobenzyl group. In some aspects, the reversible terminator nucleotide comprises a fluorescent label.

[0014] In some aspects, the photoactivatable terminator nucleotide comprises a structure of the formula:

[0015]

[0016] in:

[0017] R1 is O or S;

[0018] R2 is hydrogen or hydroxyl;

[0019] R3 is an alkyl group (C≤8) or substituted alkyl (C≤8) ;

[0020] R4 is

[0021] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0022] alkyl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0023] R5, R6 and R7 are each independently:

[0024] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0025] alkyl (C≤6) , alkenyl (C≤6) , alkynyl (C≤6) , aromatic (C≤6) , arylalkyl (C≤8) , heteroaryl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0026] A set of the following formulas:

[0027]

[0028] in

[0029] X is

[0030] -O-, -S-, or -NH-; or

[0031] Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , or a substituted form of any of these groups;

[0032] Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ;

[0033] n is an integer from 0 to 6; and

[0034] m is an integer from 0 to 6; or

[0035] - linker-reporter;

[0036] or its tautomers or optical isomers.

[0037] In some aspects, R7 is methoxy. In some aspects, the photoactivatable termination nucleotide comprises a structure of the formula:

[0038]

[0039] Wherein R5 is -linker-reporter. In some aspects, the photoactivatable termination nucleotide comprises a structure of the formula:

[0040]

[0041] In one embodiment, the present invention provides a population of oligonucleotide molecules, each of which comprises an amplification segment, a hybridization segment, and a reversible termination nucleotide from 5' to 3'. In some aspects, the amplification segment is an RNA polymerase promoter. In some aspects, the amplification segment is a primer binding site. In some aspects, the amplification segment comprises about 7 to about 50 nucleotides. In some aspects, the hybridization segment comprises a degenerate nucleotide sequence. In some aspects, each nucleic acid molecule in the population comprises a unique hybridization segment sequence. In some aspects, the hybridization segment comprises one or more known nucleotide sequences. In some aspects, each known nucleotide sequence is complementary to a target genome or mitochondrial DNA sequence. In some aspects, each known nucleotide sequence is complementary to a target RNA sequence. In some aspects, the hybridization segment comprises a poly-T sequence. In some aspects, the hybridization segment comprises about 7 to about 30 nucleotides. In some aspects, the hybridization segment comprises about 15 nucleotides. In some aspects, the oligonucleotides of the population also comprise an index barcode segment between the amplification segment and the hybridization segment. In some aspects, the oligonucleotides of the population further comprise a spacer segment located between the RNA polymerase promoter segment and the index barcode segment. In some aspects, the reversible terminator nucleotide comprises a nitrobenzyl group. In some aspects, the reversible terminator nucleotide comprises a fluorescent label.

[0042] In some aspects, the photoactivatable terminator nucleotide comprises a structure of the formula:

[0043]

[0044] in:

[0045] R1 is O or S;

[0046] R2 is hydrogen or hydroxyl;

[0047] R3 is an alkyl group (C≤8) or substituted alkyl (C≤8) ;

[0048] R4 is

[0049] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0050] alkyl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0051] R5, R6 and R7 are each independently:

[0052] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0053] alkyl (C≤6) , alkenyl (C≤6) , alkynyl (C≤6) , aromatic (C≤6) , arylalkyl (C≤8) , heteroaryl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0054] A set of the following formulas:

[0055]

[0056] in

[0057] X is

[0058] -O-, -S-, or -NH-; or

[0059] Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , or a substituted form of any of these groups;

[0060] Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ;

[0061] n is an integer from 0 to 6; and

[0062] m is an integer from 0 to 6; or

[0063] - linker-reporter;

[0064] or its tautomers or optical isomers.

[0065] In some aspects, R7 is methoxy. In some aspects, the photoactivatable termination nucleotide comprises a structure of the formula:

[0066]

[0067] Wherein R5 is -linker-reporter. In some aspects, the photoactivatable termination nucleotide comprises a structure of the formula:

[0068]

[0069] In one embodiment, provided herein is a method for identifying open DNA regions in a cell, the method comprising: (a) introducing a population of oligonucleotide molecules into the cell, wherein each molecule comprises, from 5′ to 3′, an amplification segment, an index barcode segment, a hybridization segment, and a reversible termination nucleotide; (b) incubating the cell under conditions that allow the hybridization segment of the population of oligonucleotide molecules to anneal to an open DNA region; (c) activating at least a portion of the annealed oligonucleotide molecules to expose an extendable 3' hydroxyl group; and (d) synthesizing cDNA from the open DNA by extending the activated oligonucleotide molecules from their extendable 3' hydroxyl groups.

[0070] In some aspects, the amplification segment is an RNA polymerase promoter. In some aspects, the amplification segment is a primer binding site. In some aspects, the amplification segment comprises about 7 to about 50 nucleotides. In some aspects, the hybridization segment of the oligonucleotide population comprises a degenerate nucleotide sequence. In some aspects, each nucleic acid molecule in the population comprises a unique hybridization segment sequence. In some aspects, the hybridization segment comprises one or more known nucleotide sequences. In certain aspects, each known nucleotide sequence is complementary to a target genome or mitochondrial DNA sequence. In some aspects, the hybridization segment comprises about 7 to about 30 nucleotides. In some aspects, the hybridization segment comprises about 15 nucleotides. In some aspects, the oligonucleotides of the population also comprise a spacer segment between the amplification segment and the index barcode segment. In some aspects, the photoactivatable termination nucleotide comprises a structure of the following formula:

[0071]

[0072] in:

[0073] R1 is O or S;

[0074] R2 is hydrogen or hydroxyl;

[0075] R3 is an alkyl group (C≤8) or substituted alkyl (C≤8) ;

[0076] R4 is

[0077] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0078] alkyl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide(C≤6) , or a substituted form of any of these groups;

[0079] R5, R6 and R7 are each independently:

[0080] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0081] alkyl (C≤6) , alkenyl (C≤6) , alkynyl (C≤6) , aromatic (C≤6) , arylalkyl (C≤8) , heteroaryl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0082] A set of the following formulas:

[0083]

[0084] in

[0085] X is

[0086] -O-, -S-, or -NH-; or

[0087] Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , or a substituted form of any of these groups;

[0088] Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ;

[0089] n is an integer from 0 to 6; and

[0090] m is an integer from 0 to 6; or

[0091] - linker-reporter;

[0092] or its tautomers or optical isomers.

[0093] In some aspects, R7 is methoxy. In some aspects, the photoactivatable termination nucleotide comprises a structure of the formula:

[0094]

[0095] Wherein R5 is -linker-reporter. In some aspects, the photoactivatable termination nucleotide comprises a structure of the formula:

[0096]

[0097] In some aspects, the population of oligonucleotide molecules introduced in process (a) is a population of oligonucleotide molecules according to any of the embodiments of the present invention.

[0098] In some aspects, the cell is fixed. In some aspects, activation includes photoactivation. In some aspects, open DNA is open chromatin, and activation includes exposing the nucleus to ultraviolet light. In some aspects, activation is performed in the entire nucleus. In some aspects, open DNA is open mitochondrial DNA, and activation includes exposing at least one mitochondria in the cell to ultraviolet light. In some aspects, activation is performed on more than one mitochondria in the cell. In some aspects, activation is performed at a specific site in the nucleus or mitochondria. In some aspects, a specific site is identified based on the location of a target gene. In some aspects, a specific site is a transcription start site of a target gene. In some aspects, in situ hybridization is used to locate a specific site. In some aspects, activation at a specific site includes exposing the specific site to multiphoton excitation based on an in situ hybridization signal.

[0099] In some aspects, synthesizing cDNA includes adding a DNA-dependent DNA polymerase. In some aspects, the method also includes processing the synthesized cDNA to produce a double-stranded cDNA, the double-stranded cDNA comprising the index barcode segment and the amplification segment of the oligonucleotide. In some aspects, the method also includes amplifying the double-stranded cDNA. In some aspects, amplification includes PCR, rolling circle amplification, or RNA amplification. In some aspects, the method also includes obtaining a sequence of at least a portion of the double-stranded cDNA. In some aspects, the method also includes comparing the sequence with a genome or mitochondrial sequence to identify an open DNA region.

[0100] In some aspects, the method is a multiplex method, wherein the method is performed sequentially on two or more cells in a sample. In some aspects, the method is a multiplex method, wherein the method is performed sequentially on two or more specific sites in the cells. In certain aspects, the population of oligonucleotide molecules introduced during each round of composite amplification comprises a unique index barcode segment.

[0101] In some aspects, the method is a method of classifying a cell. In some aspects, the method is a method of predicting or determining a subtype of a cell.

[0102] In some aspects, the method further includes determining whether the open DNA region is transcriptionally active, wherein the method further includes, after process (d): (e) incubating the cell under conditions that substantially allow only unextended oligonucleotides to denature from the open DNA; (f) inactivating or removing the denatured unextended oligonucleotides; (g) introducing a second population of oligonucleotide molecules into the cell, wherein each molecule comprises, from 5′ to 3′, an amplification segment, an index barcode segment different from the index barcode segment of the oligonucleotide molecule introduced in process (a), a hybridization segment, and a reversible termination nucleotide; (h) incubating the cell under conditions that allow the hybridization segment of the population of oligonucleotide molecules to anneal to the expressed RNA; (i) activating at least a portion of the annealed oligonucleotide molecules to expose an extendable 3′ hydroxyl group; and (j) synthesizing cDNA from the expressed RNA by extending the activated oligonucleotide molecules from their extendable 3′ hydroxyl groups.

[0103] In some aspects, the amplification segment is an RNA polymerase promoter. In some aspects, the amplification segment is a primer binding site. In some aspects, the amplification segment comprises about 7 to about 50 nucleotides. In some aspects, the hybridization segment of the second oligonucleotide molecule population comprises a poly-T sequence. In some aspects, the hybridization segment of the second oligonucleotide molecule population comprises one or more known nucleotide sequences. In certain aspects, each known nucleotide sequence is complementary to the target RNA sequence.

[0104] In some aspects, the hybridization segment of the second population of oligonucleotide molecules comprises about 7 to about 30 nucleotides. In some aspects, the hybridization segment of the second population of oligonucleotide molecules comprises about 15 nucleotides. In some aspects, the oligonucleotides of the population further comprise a spacer segment between the amplification segment and the index barcode segment. In some aspects, the photoactivatable termination nucleotide comprises a structure of the formula:

[0105]

[0106] in:

[0107] R1 is O or S;

[0108] R2 is hydrogen or hydroxyl;

[0109] R3 is an alkyl group (C≤8) or substituted alkyl (C≤8) ;

[0110] R4 is

[0111] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0112] alkyl (C≤6) , acyl(C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0113] R5, R6 and R7 are each independently:

[0114] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0115] alkyl (C≤6) , alkenyl (C≤6) , alkynyl (C≤6) , aromatic (C≤6) , arylalkyl (C≤8) , heteroaryl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0116] A set of the following formulas:

[0117]

[0118] in

[0119] X is

[0120] -O-, -S-, or -NH-; or

[0121] Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , or a substituted form of any of these groups;

[0122] Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ;

[0123] n is an integer from 0 to 6; and

[0124] m is an integer from 0 to 6; or

[0125] - linker-reporter;

[0126] or its tautomers or optical isomers.

[0127] In some aspects, R7 is methoxy. In some aspects, the photoactivatable termination nucleotide comprises a structure of the formula:

[0128]

[0129] Wherein R5 is -linker-reporter. In some aspects, the photoactivatable termination nucleotide comprises a structure of the formula:

[0130]

[0131] In some aspects, the second population of oligonucleotide molecules introduced in process (g) is a population of oligonucleotide molecules according to any of the embodiments of the present invention.

[0132] In some aspects, the cell is fixed. In some aspects, activation includes photoactivation. In some aspects, activation includes exposing the cytoplasm to ultraviolet light. In some aspects, activation is performed throughout the cytoplasm. In some aspects, activation is performed at a specific site in the cytoplasm. In some aspects, the specific site is an axon or a dendrite. In some aspects, synthesizing cDNA includes adding RNA-dependent DNA polymerase. In some aspects, the method also includes processing the synthesized cDNA to produce double-stranded cDNA, and the double-stranded cDNA includes the index barcode segment and the promoter region segment of the oligonucleotide. In some aspects, the method also includes amplifying the double-stranded cDNA. In some aspects, amplification includes PCR, rolling circle amplification, or RNA amplification. In some aspects, the method also includes obtaining a sequence of at least a portion of the double-stranded cDNA to identify the expressed RNA.

[0133] In some aspects, the method is a multiplex method, wherein the method is performed sequentially on two or more specific sites in the cell. In some aspects, the method is a multiplex method, wherein the method is performed sequentially on two or more cells in a sample. In certain aspects, the population of oligonucleotide molecules introduced during each round of composite amplification comprises a unique index barcode segment.

[0134] In one embodiment, provided herein is a method for identifying RNA expressed in a cell, the method comprising: (a) introducing a population of oligonucleotide molecules into the cell, wherein each molecule comprises, from 5′ to 3′, an amplification segment, an index barcode segment, a hybridization segment, and a reversible termination nucleotide; (b) incubating the cell under conditions that allow the hybridization segment of the population of oligonucleotide molecules to anneal to the expressed RNA; (c) activating at least a portion of the annealed oligonucleotide molecules to expose an extendable 3′ hydroxyl group; and (d) synthesizing cDNA from the expressed RNA by extending the activated oligonucleotide molecules from their extendable 3′ hydroxyl groups.

[0135] In some aspects, the amplification segment is an RNA polymerase promoter. In some aspects, the amplification segment is a primer binding site. In some aspects, the amplification segment comprises about 7 to about 50 nucleotides. In some aspects, the hybridization segment comprises a poly-T sequence. In some aspects, the hybridization segment comprises one or more known nucleotide sequences. In some aspects, each known nucleotide sequence is complementary to the target RNA sequence. In some aspects, the hybridization segment comprises about 7 to about 30 nucleotides. In some aspects, the hybridization segment comprises about 15 nucleotides. In some aspects, the oligonucleotides of the population further comprise a spacer segment between the amplification segment and the index barcode segment. In some aspects, the photoactivatable termination nucleotide comprises a structure of the following formula:

[0136]

[0137]

[0138] in:

[0139] R1 is O or S;

[0140] R2 is hydrogen or hydroxyl;

[0141] R3 is an alkyl group (C≤8) or substituted alkyl (C≤8) ;

[0142] R4 is

[0143] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0144] alkyl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0145] R5, R6 and R7 are each independently:

[0146] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0147] alkyl (C≤6) , alkenyl (C≤6) , alkynyl (C≤6) , aromatic (C≤6) , arylalkyl (C≤8) , heteroaryl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0148] A set of the following formulas:

[0149]

[0150] in

[0151] X is

[0152] -O-, -S-, or -NH-; or

[0153] Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , or a substituted form of any of these groups;

[0154] Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ;

[0155] n is an integer from 0 to 6; and

[0156] m is an integer from 0 to 6; or

[0157] - linker-reporter;

[0158] or its tautomers or optical isomers.

[0159] In some aspects, R7 is methoxy. In some aspects, the photoactivatable termination nucleotide comprises a structure of the formula:

[0160]

[0161] Wherein R5 is -linker-reporter. In some aspects, the photoactivatable termination nucleotide comprises a structure of the formula:

[0162]

[0163] In some aspects, the population of oligonucleotide molecules introduced in process (a) is a population of oligonucleotide molecules according to any of the embodiments of the present invention.

[0164] In some aspects, the cell is fixed. In some aspects, activation includes photoactivation. In some aspects, activation includes exposing the cytoplasm to ultraviolet light. In some aspects, activation is performed throughout the cytoplasm. In some aspects, activation is performed at a specific site in the cytoplasm. In some aspects, the specific site is an axon or a dendrite.

[0165] In some aspects, synthesizing cDNA includes adding RNA-dependent DNA polymerase. In some aspects, the method also includes processing the synthesized cDNA to produce double-stranded cDNA, the double-stranded cDNA comprising the index barcode segment and the amplification segment of the oligonucleotide. In some aspects, the method also includes amplifying the double-stranded cDNA. In some aspects, amplification includes PCR, rolling circle amplification, or RNA amplification. In some aspects, the method also includes obtaining a sequence of at least a portion of the double-stranded cDNA to identify the expressed RNA.

[0166] In some aspects, the method is a multiplex method, wherein the method is performed sequentially on two or more specific sites in the cell. In some aspects, the method is a multiplex method, wherein the method is performed sequentially on two or more cells in a sample. In certain aspects, the population of oligonucleotide molecules introduced during each round of composite amplification comprises a unique index barcode segment.

[0167] In one embodiment, provided herein is a kit comprising a population of oligonucleotides according to any one of the embodiments of the present invention, and at least one of a DNA polymerase, an RNA polymerase, a reverse transcriptase, a deoxyribonucleotide and / or a ribonucleotide.

[0168] The following numbered paragraphs describe additional and / or alternative aspects of the invention:

[0169] 1. An oligonucleotide molecule comprising, from 5' to 3', an amplification segment, a hybridization segment and a reversible termination nucleotide.

[0170] 2. The oligonucleotide molecule according to paragraph 1, wherein the amplification segment is an RNA polymerase promoter.

[0171] 3. The oligonucleotide molecule according to paragraph 1, wherein the amplification segment is a primer binding site.

[0172] 4. The oligonucleotide molecule according to any one of paragraphs 1-3, wherein the amplification segment comprises about 7 to about 50 nucleotides.

[0173] 5. The oligonucleotide molecule according to any one of paragraphs 1-4, wherein the hybridizing segment comprises a random nucleotide sequence.

[0174] 6. The oligonucleotide molecule according to any one of paragraphs 1-4, wherein the hybridizing segment comprises a known nucleotide sequence.

[0175] 7. The oligonucleotide molecule according to paragraph 6, wherein the known nucleotide sequence is complementary to a target genomic or mitochondrial DNA sequence.

[0176] 8. The oligonucleotide molecule according to paragraph 6, wherein the known nucleotide sequence is complementary to the target RNA sequence.

[0177] 9. The oligonucleotide molecule according to any one of paragraphs 1-4, wherein the hybridizing segment comprises a poly-T sequence.

[0178] 10. The oligonucleotide molecule according to any one of paragraphs 1-9, wherein the hybridizing segment comprises about 7 to about 30 nucleotides.

[0179] 11. The oligonucleotide molecule according to any one of paragraphs 1-10, wherein the hybridizing segment comprises about 15 nucleotides.

[0180] 12. The oligonucleotide molecule according to any one of paragraphs 1-11, further comprising an index barcode segment located between the amplification segment and the hybridization segment.

[0181] 13. The oligonucleotide molecule according to paragraph 12, further comprising a spacer segment located between the amplification segment and the index barcode segment.

[0182] 14. An oligonucleotide molecule according to any one of paragraphs 1-13, wherein the reversible terminator nucleotide comprises a nitrobenzyl group.

[0183] 15. The oligonucleotide molecule according to any one of paragraphs 1-14, wherein the reversible terminator nucleotide comprises a fluorescent label.

[0184] 16. The oligonucleotide molecule according to any one of paragraphs 1-15, wherein the reversible terminator nucleotide is a photoactivatable terminator nucleotide.

[0185] 17. An oligonucleotide molecule according to paragraph 16, wherein the photoactivatable termination nucleotide comprises a structure of the following formula:

[0186]

[0187] in:

[0188] R1 is O or S;

[0189] R2 is hydrogen or hydroxyl;

[0190] R3 is an alkyl group (C≤8) or substituted alkyl (C≤8) ;

[0191] R4 is

[0192] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0193] alkyl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0194] R5, R6 and R7 are each independently:

[0195] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0196] alkyl (C≤6) , alkenyl (C≤6) , alkynyl (C≤6) , aromatic (C≤6) , arylalkyl (C≤8) , heteroaryl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0197] A set of the following formulas:

[0198]

[0199] in

[0200] X is

[0201] -O-, -S-, or -NH-; or

[0202] Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene(C≤12) , or a substituted form of any of these groups;

[0203] Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ;

[0204] n is an integer from 0 to 6; and

[0205] m is an integer from 0 to 6; or

[0206] - linker-reporter;

[0207] or its tautomers or optical isomers.

[0208] 18. The oligonucleotide molecule according to paragraph 17, wherein R7 is methoxy.

[0209] 19. An oligonucleotide molecule according to paragraph 18, wherein the photoactivatable termination nucleotide comprises a structure of the following formula:

[0210]

[0211] Wherein R5 is -linker-reporter.

[0212] 20. The oligonucleotide molecule according to paragraph 19, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

[0213]

[0214] 21. A population of oligonucleotide molecules, each oligonucleotide molecule comprising, from 5' to 3', an amplification segment, a hybridization segment and a reversible termination nucleotide.

[0215] 22. A population according to paragraph 21, wherein the amplification segment is an RNA polymerase promoter.

[0216] 23. A population according to paragraph 21, wherein the amplification segment is a primer binding site.

[0217] 24. A population according to any of paragraphs 21-23, wherein the amplified segment comprises about 7 to about 50 nucleotides.

[0218] 25. A population according to any of paragraphs 21-24, wherein the hybridization segment comprises a degenerate nucleotide sequence.

[0219] 26. A population according to any of paragraphs 21-25, wherein each nucleic acid molecule in the population comprises a unique hybridization segment sequence.

[0220] 27. A population according to any of paragraphs 21-24, wherein the hybridization segment comprises one or more known nucleotide sequences.

[0221] 28. A population according to paragraph 27, wherein each known nucleotide sequence is complementary to a target genomic or mitochondrial DNA sequence.

[0222] 29. A population according to paragraph 27, wherein each known nucleotide sequence is complementary to a target RNA sequence.

[0223] 30. A population according to any of paragraphs 21-24, wherein the hybridization segment comprises a poly-T sequence.

[0224] 31. A population according to any of paragraphs 21-30, wherein the hybridization segment comprises about 7 to about 30 nucleotides.

[0225] 32. A population according to any of paragraphs 21-31, wherein the hybridization segment comprises about 15 nucleotides.

[0226] 33. The population of any of paragraphs 21-32, further comprising an index barcode segment located between the amplification segment and the hybridization segment.

[0227] 34. The population of paragraph 33, further comprising a spacer segment between the RNA polymerase promoter segment and the index barcode segment.

[0228] 35. A population according to any of paragraphs 21-34, wherein the reversible termination nucleotide comprises a nitrobenzyl group.

[0229] 36. A population according to any of paragraphs 21-35, wherein the reversible terminator nucleotide comprises a fluorescent label.

[0230] 37. A population according to any of paragraphs 21-36, wherein the reversible terminator nucleotide is a photoactivatable terminator nucleotide.

[0231] 38. A population according to paragraph 37, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

[0232]

[0233]

[0234] in:

[0235] R1 is O or S;

[0236] R2 is hydrogen or hydroxyl;

[0237] R3 is an alkyl group (C≤8) or substituted alkyl (C≤8) ;

[0238] R4 is

[0239] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0240] alkyl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0241] R5, R6 and R7 are each independently:

[0242] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0243] alkyl (C≤6) , alkenyl (C≤6) , alkynyl (C≤6) , aromatic (C≤6) , arylalkyl (C≤8) , heteroaryl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0244] A set of the following formulas:

[0245]

[0246] in

[0247] X is

[0248] -O-, -S-, or -NH-; or

[0249] Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , or a substituted form of any of these groups;

[0250] Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ;

[0251] n is an integer from 0 to 6; and

[0252] m is an integer from 0 to 6; or

[0253] - linker-reporter;

[0254] or its tautomers or optical isomers.

[0255] 39. A population according to paragraph 38, wherein R7 is methoxy.

[0256] 40. A population according to paragraph 39, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

[0257]

[0258] Wherein R5 is -linker-reporter.

[0259] 41. A population according to paragraph 40, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

[0260]

[0261] 42. A method for identifying open DNA regions in a cell, the method comprising:

[0262] (a) introducing a population of oligonucleotide molecules into the cell, wherein each molecule comprises, from 5′ to 3′, an amplification segment, an index barcode segment, a hybridization segment, and a reversible termination nucleotide;

[0263] (b) incubating the cells under conditions that allow annealing of the hybridized segments of the population of oligonucleotide molecules to open DNA regions;

[0264] (c) activating at least a portion of the annealed oligonucleotide molecules to expose extendable 3' hydroxyl groups; and

[0265] (d) synthesizing cDNA from the open DNA by extending the activated oligonucleotide molecule from its extendable 3' hydroxyl group.

[0266] 43. The method of paragraph 42, wherein the amplification segment is an RNA polymerase promoter.

[0267] 44. The method of paragraph 42, wherein the amplification segment is a primer binding site.

[0268] 45. The method of any one of paragraphs 42-44, wherein the amplified segment comprises about 7 to about 50 nucleotides.

[0269] 46. ​​The method according to any of paragraphs 42-45, wherein the hybridizing segment of the oligonucleotide population comprises a degenerate nucleotide sequence.

[0270] 47. The method of any one of paragraphs 42-46, wherein each nucleic acid molecule in the population comprises a unique hybridization segment sequence.

[0271] 48. The method of any one of paragraphs 42-45, wherein the hybridization segment comprises one or more known nucleotide sequences.

[0272] 49. The method of paragraph 48, wherein each known nucleotide sequence is complementary to a target genomic or mitochondrial DNA sequence.

[0273] 50. The method of any one of paragraphs 42-49, wherein the hybridization segment comprises about 7 to about 30 nucleotides.

[0274] 51. The method of any one of paragraphs 42-50, wherein the hybridization segment comprises about 15 nucleotides.

[0275] 52. The method of any one of paragraphs 42-51, wherein the population of oligonucleotide molecules further comprises a spacer segment located between the amplification segment and the index barcode segment.

[0276] 53. The method according to any one of paragraphs 42-52, wherein the reversible terminator nucleotide is a photoactivatable terminator nucleotide.

[0277] 54. The method of paragraph 53, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

[0278]

[0279] in:

[0280] R1 is O or S;

[0281] R2 is hydrogen or hydroxyl;

[0282] R3 is an alkyl group (C≤8) or substituted alkyl (C≤8) ;

[0283] R4 is

[0284] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0285] alkyl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino(C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0286] R5, R6 and R7 are each independently:

[0287] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0288] alkyl (C≤6) , alkenyl (C≤6) , alkynyl (C≤6) , aromatic (C≤6) , arylalkyl (C≤8) , heteroaryl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0289] A set of the following formulas:

[0290]

[0291] in

[0292] X is

[0293] -O-, -S-, or -NH-; or

[0294] Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , or a substituted form of any of these groups;

[0295] Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ;

[0296] n is an integer from 0 to 6; and

[0297] m is an integer from 0 to 6; or

[0298] - linker-reporter;

[0299] or its tautomers or optical isomers.

[0300] 55. The method according to paragraph 54, wherein R7 is methoxy.

[0301] 56. The method of paragraph 55, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

[0302]

[0303] Wherein R5 is -linker-reporter.

[0304] 57. The method of paragraph 56, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

[0305]

[0306] 58. The method according to any one of paragraphs 42-57, wherein the population of oligonucleotide molecules introduced in process (a) is a population of oligonucleotide molecules according to any one of paragraphs 21-28 and 31-41.

[0307] 59. The method of any one of paragraphs 42-58, wherein the cells are fixed.

[0308] 60. The method of any one of paragraphs 42-59, wherein the open DNA is open chromatin, wherein activation comprises exposing the nucleus to ultraviolet light.

[0309] 61. The method of any one of paragraphs 42-60, wherein activation is performed throughout the cell nucleus.

[0310] 62. The method of any of paragraphs 42-59, wherein the open DNA is open mitochondrial DNA, wherein activation comprises exposing at least one mitochondria in the cell to ultraviolet light.

[0311] 63. The method of any one of paragraphs 42-59 and 62, wherein activation is performed on more than one mitochondria in the cell.

[0312] 64. The method of any one of paragraphs 42-60 or 62, wherein activation is performed at a specific site within the nucleus or mitochondria.

[0313] 65. The method of paragraph 64, wherein the specific site is identified based on the location of the gene of interest.

[0314] 66. The method according to paragraph 65, wherein the specific site is the transcription start site of the target gene.

[0315] 67. The method according to paragraph 65 or 66, wherein in situ hybridization is used to locate the specific site.

[0316] 68. The method of paragraph 67, wherein activation at the specific site comprises exposing the specific site to multi-photon excitation based on an in situ hybridization signal.

[0317] 69. The method of any one of paragraphs 42-68, wherein synthesizing cDNA comprises adding a DNA-dependent DNA polymerase.

[0318] 70. The method of any of paragraphs 42-69, further comprising processing the synthesized cDNA to produce a double-stranded cDNA comprising the index barcode segment and the amplification segment of the oligonucleotide.

[0319] 71. The method according to paragraph 70, further comprising amplifying the double-stranded cDNA.

[0320] 72. The method of paragraph 71, wherein amplification comprises PCR, rolling circle amplification, or RNA amplification.

[0321] 73. The method of paragraph 71 or 72, further comprising obtaining the sequence of at least a portion of the double-stranded cDNA.

[0322] 74. The method of paragraph 73, further comprising aligning the sequence with a genomic or mitochondrial sequence to identify open DNA regions.

[0323] 75. The method of any of paragraphs 42-74, wherein the method is a multiplex method, wherein the method is performed sequentially on two or more cells in a sample.

[0324] 76. The method of any one of paragraphs 42-74, wherein the method is a multiplex method, wherein the method is performed sequentially on two or more specific sites in the cell.

[0325] 77. The method of paragraph 75 or 76, wherein the population of oligonucleotide molecules introduced during each round of multiplex amplification comprises a unique index barcode segment.

[0326] 78. The method of any one of paragraphs 42-77, wherein the method is further defined as a method of classifying cells.

[0327] 79. The method according to any one of paragraphs 42-77, wherein the method is further defined as a method for predicting or determining a subtype of a cell.

[0328] 80. The method according to any one of paragraphs 42-79, wherein the method further comprises determining whether the open DNA region has transcriptional activity, wherein the method further comprises, after process (d):

[0329] (e) incubating the cells under conditions that substantially allow only unextended oligonucleotides to denature from the open DNA;

[0330] (f) inactivating or removing the denatured unextended oligonucleotide;

[0331] (g) introducing into the cell a second population of oligonucleotide molecules, wherein each molecule comprises, from 5′ to 3′, an amplification segment, an index barcode segment that is different from the index barcode segment of the oligonucleotide molecule introduced in process (a), a hybridization segment, and a reversible terminator nucleotide;

[0332] (h) incubating the cell under conditions that allow annealing of the hybridizing segments of the population of oligonucleotide molecules to the expressed RNA;

[0333] (i) activating at least a portion of the annealed oligonucleotide molecules to expose extendable 3' hydroxyl groups; and

[0334] (j) synthesizing cDNA from the expressed RNA by extending the activated oligonucleotide molecule from its extendable 3' hydroxyl group.

[0335] 81. The method of paragraph 80, wherein the amplification segment is an RNA polymerase promoter.

[0336] 82. The method of paragraph 80, wherein the amplification segment is a primer binding site.

[0337] 83. The method of any one of paragraphs 80-82, wherein the amplified segment comprises about 7 to about 50 nucleotides.

[0338] 84. The method according to any one of paragraphs 80-83, wherein the hybridization segment of the second population of oligonucleotide molecules comprises a poly-T sequence.

[0339] 85. The method according to any one of paragraphs 80-84, wherein the hybridizing segments of the second population of oligonucleotide molecules comprise one or more known nucleotide sequences.

[0340] 86. The method of paragraph 85, wherein each known nucleotide sequence is complementary to a target RNA sequence.

[0341] 87. The method according to any one of paragraphs 80-86, wherein the hybridization segment of the second population of oligonucleotide molecules comprises about 7 to about 30 nucleotides.

[0342] 88. The method according to any one of paragraphs 80-87, wherein the hybridizing segment of the second population of oligonucleotide molecules comprises about 15 nucleotides.

[0343] 89. The method of any one of paragraphs 80-88, wherein the population of oligonucleotide molecules further comprises a spacer segment located between the amplification segment and the index barcode segment.

[0344] 90. The method according to any one of paragraphs 80-89, wherein the reversible terminator nucleotide is a photoactivatable terminator nucleotide.

[0345] 91. The method of paragraph 90, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

[0346]

[0347] in:

[0348] R1 is O or S;

[0349] R2 is hydrogen or hydroxyl;

[0350] R3 is an alkyl group (C≤8) or substituted alkyl (C≤8) ;

[0351] R4 is

[0352] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0353] alkyl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0354] R5, R6 and R7 are each independently:

[0355] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0356] alkyl (C≤6) , alkenyl (C≤6) , alkynyl (C≤6) , aromatic (C≤6) , arylalkyl (C≤8) , heteroaryl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy(C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0357] A set of the following formulas:

[0358]

[0359] in

[0360] X is

[0361] -O-, -S-, or -NH-; or

[0362] Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , or a substituted form of any of these groups;

[0363] Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ;

[0364] n is an integer from 0 to 6; and

[0365] m is an integer from 0 to 6; or

[0366] - linker-reporter;

[0367] or its tautomers or optical isomers.

[0368] 92. The method of paragraph 91, wherein R7 is methoxy.

[0369] 93. The method of paragraph 92, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

[0370]

[0371] Wherein R5 is -linker-reporter.

[0372] 94. The method of paragraph 93, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

[0373]

[0374] 95. The method according to any one of paragraphs 80-94, wherein the second population of oligonucleotide molecules introduced in process (g) is a population of oligonucleotide molecules according to any one of paragraphs 21-27 and 29-41.

[0375] 96. The method of any one of paragraphs 80-95, wherein the cells are fixed.

[0376] 97. The method of any of paragraphs 80-96, wherein activation comprises exposing the cytoplasm to ultraviolet light.

[0377] 98. The method of any of paragraphs 80-97, wherein activation is performed throughout the cytoplasm.

[0378] 99. The method of any one of paragraphs 80-97, wherein activation is performed at a specific site within the cytoplasm.

[0379] 100. The method of paragraph 99, wherein the specific site is an axon or a dendrite.

[0380] 101. The method of any of paragraphs 80-100, wherein synthesizing cDNA comprises adding an RNA-dependent DNA polymerase.

[0381] 102. The method of any of paragraphs 80-101, further comprising processing the synthesized cDNA to produce a double-stranded cDNA comprising the index barcode segment and the promoter region segment of the oligonucleotide.

[0382] 103. The method according to paragraph 102 further comprises amplifying the double-stranded cDNA.

[0383] 104. The method of paragraph 103, wherein amplification comprises PCR, rolling circle amplification, or RNA amplification.

[0384] 105. The method of paragraph 103 or 104, further comprising obtaining the sequence of at least a portion of the double-stranded cDNA to thereby identify the expressed RNA.

[0385] 106. The method of any one of paragraphs 99-105, wherein the method is a multiplex method, wherein the method is performed sequentially on two or more specific sites in the cell.

[0386] 107. The method of any of paragraphs 83-106, wherein the method is a multiplex method, wherein the method is performed sequentially on two or more cells in a sample.

[0387] 108. The method of paragraph 106 or 107, wherein the population of oligonucleotide molecules introduced during each round of multiplex amplification comprises a unique index barcode segment.

[0388] 109. A method for identifying RNA expressed in a cell, the method comprising:

[0389] (a) introducing a population of oligonucleotide molecules into the cell, wherein each molecule comprises, from 5′ to 3′, an amplification segment, an index barcode segment, a hybridization segment, and a reversible termination nucleotide;

[0390] (b) incubating the cell under conditions that allow annealing of the hybridizing segments of the population of oligonucleotide molecules to the expressed RNA;

[0391] (c) activating at least a portion of the annealed oligonucleotide molecules to expose extendable 3' hydroxyl groups; and

[0392] (d) synthesizing cDNA from the expressed RNA by extending the activated oligonucleotide molecule from its extendable 3' hydroxyl group.

[0393] 110. The method of paragraph 109, wherein the amplification segment is an RNA polymerase promoter.

[0394] 111. The method of paragraph 109, wherein the amplification segment is a primer binding site.

[0395] 112. The method of any one of paragraphs 109-111, wherein the amplified segment comprises about 7 to about 50 nucleotides.

[0396] 113. The method of any one of paragraphs 109-112, wherein the hybridization segment comprises a poly-T sequence.

[0397] 114. The method of any one of paragraphs 109-112, wherein the hybridization segment comprises one or more known nucleotide sequences.

[0398] 115. The method of paragraph 114, wherein each known nucleotide sequence is complementary to a target RNA sequence.

[0399] 116. The method of any one of paragraphs 109-115, wherein the hybridization segment comprises about 7 to about 30 nucleotides.

[0400] 117. The method of any one of paragraphs 109-116, wherein the hybridization segment comprises approximately 15 nucleotides.

[0401] 118. The method according to any one of paragraphs 109-117, wherein the population of oligonucleotide molecules further comprises a spacer segment located between the amplification segment and the index barcode segment.

[0402] 119. The method according to any one of paragraphs 109-118, wherein the reversible terminator nucleotide is a photoactivatable terminator nucleotide.

[0403] 120. The method of paragraph 119, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

[0404]

[0405]

[0406] in:

[0407] R1 is O or S;

[0408] R2 is hydrogen or hydroxyl;

[0409] R3 is an alkyl group (C≤8) or substituted alkyl (C≤8) ;

[0410] R4 is

[0411] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0412] alkyl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0413] R5, R6 and R7 are each independently:

[0414] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0415] alkyl (C≤6) , alkenyl (C≤6) , alkynyl (C≤6) , aromatic (C≤6) , arylalkyl (C≤8) , heteroaryl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0416] A set of the following formulas:

[0417]

[0418] in

[0419] X is

[0420] -O-, -S-, or -NH-; or

[0421] Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , or a substituted form of any of these groups;

[0422] Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ;

[0423] n is an integer from 0 to 6; and

[0424] m is an integer from 0 to 6; or

[0425] - linker-reporter;

[0426] or its tautomers or optical isomers.

[0427] 121. The method of paragraph 120, wherein R7 is methoxy.

[0428] 122. The method of paragraph 121, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

[0429]

[0430] Wherein R5 is -linker-reporter.

[0431] 123. The method of paragraph 122, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

[0432]

[0433] 124. The method according to any one of paragraphs 109-123, wherein the population of oligonucleotide molecules introduced in process (a) is a population of oligonucleotide molecules according to any one of paragraphs 21-27 and 29-41.

[0434] 125. The method of any of paragraphs 109-124, wherein the cells are fixed.

[0435] 126. The method of any of paragraphs 109-125, wherein activation comprises exposing the cytoplasm to ultraviolet light.

[0436] 127. The method of any of paragraphs 109-126, wherein activation is performed throughout the cytoplasm.

[0437] 128. The method of any one of paragraphs 109-126, wherein activation is performed at a specific site within the cytoplasm.

[0438] 129. The method of paragraph 128, wherein the specific site is an axon or a dendrite.

[0439] 130. The method of any of paragraphs 109-129, wherein synthesizing cDNA comprises adding an RNA-dependent DNA polymerase.

[0440] 131. The method of any of paragraphs 105-130, further comprising processing the synthesized cDNA to produce a double-stranded cDNA comprising the index barcode segment and the amplification segment of the oligonucleotide.

[0441] 132. The method of paragraph 131, further comprising amplifying the double-stranded cDNA.

[0442] 133. The method of paragraph 132, wherein amplification comprises PCR, rolling circle amplification, or RNA amplification.

[0443] 134. The method of paragraph 132 or 133, further comprising obtaining the sequence of at least a portion of the double-stranded cDNA to thereby identify the expressed RNA.

[0444] 135. The method of any one of paragraphs 109-134, wherein the method is a multiplex method, wherein the method is performed sequentially on two or more specific sites in the cell.

[0445] 136. The method of any of paragraphs 109-135, wherein the method is a multiplex method, wherein the method is performed sequentially on two or more cells in a sample.

[0446] 137. The method of paragraph 135 or 136, wherein the population of oligonucleotide molecules introduced during each round of multiplex amplification comprises a unique index barcode segment.

[0447] 138. A kit comprising a population of oligonucleotides according to any of paragraphs 21-41, and at least one of a DNA polymerase, an RNA polymerase, a reverse transcriptase, a deoxyribonucleotide, and a ribonucleotide.

[0448] According to the following detailed description, other objects, features and advantages of the present invention will become apparent. However, it should be understood that the detailed description and specific examples, although indicating preferred embodiments of the present invention, are given by way of illustration only, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art based on the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0449] The following drawings form part of this specification and are included to further illustrate certain aspects of the present invention. The present invention may be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments given herein.

[0450] Figure 1 .Exemplary CHeX-seq multifunctional oligonucleotide (SEQ ID NO: 1). At the 3' end of the oligonucleotide there is a photoactivatable non-extendable nucleotide containing a Cy5 fluorescent moiety. Upon photoactivation, the Cy5 fluorescence disappears and a free 3'-hydroxyl group is formed, allowing the oligonucleotide to be extended in situ, thereby providing for DNA synthesis.

[0451] Figure 2 . Schematic representation of the CHeX-seq protocol for assaying transcriptionally active chromatin.

[0452] FIG. 3A to FIG. 3B Schematic diagram of two exemplary CHeX-seq oligonucleotide synthesis methods. Figure 3A The complete CHeX-seq probe (T7-BC1-N(15)-T-LTdU-Cy5) sequence is shown at the top. The oligonucleotide T7BC1-15N-T and its reverse complementary oligonucleotide T7BC1-15N-T-RC containing the T7 promoter site, Illumina 6bp barcode 1 (BC1, blue) and 15bp degenerate sequence were synthesized and annealed to each other to produce a double-stranded oligonucleotide. The Cy5-labeled light terminator (Lightning Terminator) LTdU-Cy5 was bound to the 3' end of the double-stranded oligonucleotide. The single-stranded CHeX-seq probe was harvested after double-stranded probe denaturation and HPLC purification. T7BC1-15-NT is SEQ ID NO:2, and T7BC1-15N-RC is SEQ ID NO:3. Figure 3B —T7BC1 is SEQ ID NO:4; T7BC1-RC is SEQ ID NO:5; 15N-T is SEQ ID NO:6; and A-15N-RC is SEQ ID NO:7. The sequences of the ligated pairs are provided by SEQ ID NO:2 and SEQ ID NO:3.

[0453] Figure 4 . Schematic diagram of an exemplary CHeX-seq oligonucleotide synthesis method.

[0454] Figure 5 .Exemplary fluorescently labeled photoactivatable terminator nucleotide compounds.

[0455] Figure 6 . Photoactivation at specific sites in the nucleus of a cell. To illustrate the ability of local activation in the nucleus of a cell, the nucleus was stained with antibodies against transcription factors localized in the nucleus. A specific site (dashed box) was activated by 405nm laser light, indicating that such sites (similar to HCR in situ hybridization sites) can be photoaddressed.

[0456] Figure 7 Automated cell compartment analysis based on immunofluorescence images for optical cleaning. Raw images are separated into immunofluorescence signals of selective compartments, where Tau for axons, MAP2 for dendrites, and DAPI for nuclei. Each compartment image is calculated as a binary image to automatically quantify the mean intensity and regional distribution pattern of each pixel. Somatic compartments are calculated based on DAPI / MAP2 signals and cell morphology outlines.

[0457] Figure 8 Partial correlation of the F statistic across species, controlling for gene expression levels in cortical (left) and hippocampal (right) pyramidal neurons. The axes are measures of variation, controlling for gene expression levels. “ρ” denotes partial correlation coefficient. p-values ​​are from two-sided T-tests of association. Marginal histograms are shown overlaid with normal curves.

[0458] Fig. 9 Model of chromatin variation. The signal is received by the neuron in the top panel, which activates a second messenger system (green dots), which activates the cell nucleus. Each neuron has a different arrangement of chromosomes in the nucleus (middle panel), which activate gene transcription to varying degrees when receiving a vector signal (lower panel).

[0459] Fig.10 .CHeX-seq preliminary data. Reads are quantified relative to genomic feature start and end positions for positive and control samples: transcription start sites, gene coding regions, 3'UTRs, and intergenic regions. Single cell samples are shown as separate traces and are also "merged" into a single trace. Pooled samples are shown as separate traces.

[0460] Fig.11. Annealing and activation of CHeX-seq oligonucleotides in fixed mouse cortical brain slices. 130 μm thick slices were fixed with 4% paraformaldehyde for 10 min. GFAP and Map2 antibodies were applied overnight. Fluorescently labeled species-specific secondary antibodies were added after this. The slices were then incubated with 170 nM CHeX-seq oligonucleotides for 60 min. After washing, individual cells with GFAP or Map2 staining were irradiated with a 405 laser line at 80% power. cDNA in situ synthesis was initiated after this by adding DNA polymerase and synthesis buffer. Chicken polyclonal anti-GFAP antibody (Abcam, number ab4674). Rabbit monoclonal anti-Map2 antibody was a gift from Craig Garner, Stanford. Scale bar = 20 μm, neuronal images are 2x magnification and glial cells are 1x magnification. Two mags are shown to highlight the specificity of CHeX-seq activation.

[0461] Fig.12 Schematic diagram of CHeX-seq enabled 3D chromatin niche structure analysis around a specific gene. The illustrated method will allow identification of chromatin regulatory sites that regulate expression of a specific gene. The genomic location of the gene of interest will be identified by in situ hybridization using HCR. This will provide a beacon on which to focus the CheX-seq oligonucleotide activation laser so that only CheX-seq oligonucleotides close to the FISH probe signal site will be activated.

[0462] FIG. 13A to FIG. 13G .K562 CHeX-seq benchmark. Fig.13A —Schematic diagram of the CheX-seq assay principle. Fig. 13B —CHeX-seq probes loaded into K562 cell nuclei (DIC image) and fluorescence signals in individual cell nuclei before and after activation of CHeX-seq probes (red arrows), Scale bar = 20 μm. Fig. 13C — Statistics of CHeX-seq priming sites on genomic features. Fig.13D —TSS proximal (+ / -5kb) coverage of K562 samples (all positive samples combined). Fig.13E — Z-scored coverage of TSS-proximal (upper) and CDS (lower) at the single-cell level. Fig.13F —Overlap between genes primed by CheX-seq (whole gene body > 0) and genes expressed by RNA-seq (exons > median). Figure 13G —GO functional enrichment results of CheX-RNA overlapping genes (top 20) ( Fig.13F , left side).

[0463] FIG. 14A to FIG. 14B.Genomic comparison of CHeX-seq with other open chromatin assays. Fig.14A —UCSC Genome Browser track view comparing coverage of CHeX-seq (purple) versus ATAC-seq (red), DNase-seq (blue), and FAIL-seq (green) at the OTUD5 locus. Below the four assays are regulatory interaction tracks (GeneCards Gene TSS, Enhancers, and Promoters, and GeneHancer Proximal-Distal Interactions) derived from the GeneHancer database (Fishilevich et al., 2017). The last four tracks are the transcriptome and three histone marks (H3K27ac, H3K4me1, H3K4me3s). A regulatory interaction between the promoter of OTUD5 and one of its 3′ introns is shared by all four open chromatin assays (blue rectangles). Fig. 14B —Hierarchical clustering of open chromatin assays, transcriptome, and epigenome at 10 kb bin (left) and 50 kb bin (right) resolution using binarized coverage and Jaccard distance.

[0464] Figures 15A-15C. Correlation of distance of CHeX-seq reads from TSS with gene RNA abundance. Figure 15A—Bulk K562 RNA-seq. Figure 15B—Bulk K562 GRO-seq. Figure 15C—K562 scRNA-seq, averaged single cells. y-axis: gene expression; x-axis: distance from CHeX initiation site to TSS.

[0465] FIG. 16A to FIG. 16B .CHeX-seq chain type: detect the chain type of open chromatin. Fig.16A —Schematic diagram illustrating the assumption that CHeX-seq primed extension products should have the opposite strand type to the sense strand mRNA transcript. Fig. 16B -test Fig.16A x-axis: various genomic features used to count and binarize CHeX-seq priming events; y-axis: ratio of the number of antisense to sense CHeX-seq products.

[0466] FIG. 17A to FIG. 17F .CHeX-seq analysis of single neurons in fixed mouse tissue sections and dispersed cell cultures. Fig.17A —Schematic diagram of CHeX-seq analysis of paraformaldehyde-fixed tissue sections. Fig. 17B— Hippocampal slice showing neurons immunolabeled for MAP2 (green). Red fluorescence indicates localization of the CHeX-seq probe. The rightmost panel shows reduced fluorescence signal in activated individual neuronal nuclei (white arrows); scale bar = 20 μm. Fig. 17C - Paraformaldehyde-fixed cultured cortical neurons (left panel) and nuclear fluorescence of CHeX-seq primers (middle panel) shown by DIC microscopy. After probe activation, the signal decreases (right panel; quantified in the inset of the right panel); Scale bar = 20 μm. Fig.17D —CheX transcriptome comparison. Left, mouse fixed tissue sections; right, mouse scattered neurons. Rows are scRNA-seq average expression in exonic or intronic regions, and columns are CHeX-seq binarized priming signals in the entire gene body, exonic or intronic regions. Fig.17E —Correlation between CHeX-seq priming frequency and transcriptional activity in intronic regions in hippocampal slices. Fig.17F —Correlation between CHeX-seq priming frequency and transcriptional variability in intronic regions in mouse tissue sections.

[0467] Fig.18 .Chromosome landscape of single-stranded open chromatin across cell types. Distribution of CHeX initiation sites on chromosomes; color: fractional initiation frequency for each chromosome. (left panel) mouse astrocyte culture, (middle panel) mouse dispersed neuronal cells, (right panel) mouse neuronal slices.

[0468] Fig.19 Schematic representation of the CHeX-seq aRNA amplification protocol. When the CHeX-seq probe T7-BC1-N(15)-T-LTdU-Cy5 is applied to PFA-fixed, Triton X-100-permeabilized cells, the degenerate N(15) sequence hybridizes to single-stranded nucleosome-depleted genomic DNA found in regions of open chromatin. First-strand DNA synthesis is initiated by DNA polymerase I following light-mediated photocleavage of the terminator group of the CHeX oligonucleotide. Following poly(G) tailing of the 3′ end of the first-strand DNA, a custom App-RC-polyC primer (Table 1) is used to prime and synthesize the second-strand DNA. Finally, RNA is amplified using linear in vitro transcription from a T7 RNA polymerase promoter incorporated into the double-stranded DNA. Round 2 first- and second-strand DNAs are then synthesized and amplified by PCR.

[0469] FIG. 20A to FIG. 20B In situ hybridization to region 630737-633960 of chromosome 1 (hg38). Fig. 20A—UCSC Genome Browser track view of a portion of chromosome 1. The CheX-seq track is similar to the ATAC-seq track, indicating that this chromosomal region is open. This is different from the DNase-seq and FAIL-seq data. Fig. 20B - The left panel is DAPI staining of K562 cell nuclei. The right panel shows fluorescence in situ hybridization signals using 8 fluorescently labeled oligonucleotides. These data show highly specific trisomy of chromosome 1 in the nuclei of K562 cells. Scale bar = 20 μm.

[0470] Fig.21 . Correlation between distance of CHeX-seq reads from TSS and RNA abundance in neurons and astrocytes.

[0471] FIG. 22A to FIG. 22F Chex-seq reads mapped to transcription start sites in K562 cells, scattered neurons and astrocytes from human and mouse, and neurons mapped in mouse brain sections. Fig.22A —K562. Fig. 22B —Human astrocytes. Fig. 22C —Human neurons. Fig.22D —Mouse astrocytes. Fig.22E —Mouse neurons. Fig.22F —Mouse neurons in situ.

[0472] FIG. 23A to FIG. 23B .CHeX-seq applied to primary astrocyte cultures from mouse and human samples. Fig.23A — Top is an image of human astrocytes, bottom is an image of mouse astrocytes. DIC (left) and DAPI images before CHeX-seq probe activation (left and middle panels, respectively), and DAPI images after activation (right panel; quantification of DAPI signal in inset). Scale bar = 20 μm. Fig. 23B — Quantification of CHeX-seq primed sites associated with genomic features in astrocytes (left) and neurons (right). Key to CHeX-seq read sites relative to the location of genomic structures. DETAILED DESCRIPTION

[0473] The process of RNA transcription requires that the cell's genomic DNA is in an open chromatin conformation, in which there is less nucleosome packaging, so that transcriptional regulatory proteins can bind and function. Similarly, mitochondrial DNA must be in an open conformation for transcription to occur. It is clear that chromatin structure is dynamic and regulated by multiple factors, including development, stress, and pharmacological attack (Fullard et al., 2017; Kozlenkov et al., 2014; Kozlenkov et al., 2016). Most chromatin modeling studies rely on the use of multiple cells to generate genomic DNA / chromatin for analysis. Chromatin analysis procedures include DnA enzyme-seq, FAIL-seq, and ChIP-seq, as well as other methods. Recently, these methods have been extended to single cells (Cusanovich et al., 2015; Buenrostro et al., 2015; Rotem et al., 2015; Clark et al., 2018). For example, the recent ATAC-seq method for mapping chromatin in single cells utilizes an assay for detecting transposase-accessible chromatin (Buenrostro et al., 2013). This method uses the Tn5 transposase to label and purify accessible nucleosome-free double-stranded DNA regions in the genome. Each of these procedures has specific advantages and disadvantages, the most important of which is that they all assess chromatin in nuclei isolated from the tissue of interest, thereby losing spatial location information and cellular microenvironmental context. To overcome these issues, CHeX-seq ( dye Color quality Violence dew( Ch romatin eX posed)) to assess chromatin conformation in fixed single cells, including neurons and astrocytes.

[0474] CHeX-seq is complementary to ATAC-seq because CHeX-seq queries single-stranded DNA, while ATAC-seq assesses double-stranded DNA. Open chromatin consists of both double-stranded DNA and single-stranded DNA (Bjursell et al., 1979; Scheer et al., 1987; Kouzine et al., 2017). The open state of chromatin is necessary for many cellular functions, such as replication, homologous recombination, DNA repair, and transcription. Although the open state of chromatin is necessary for transcription to occur, "open" may not be directly related to transcription because other trans-acting factors are also necessary (Yu et al., 2017). Single-stranded DNA is necessary for transcription in the form of a single-stranded "transcription bubble", which is reported to be about 200 bases large (Barnes et al., 2015; Bieberstein et al., 2012). Furthermore, consistent with transcription bubbles, transcriptionally active chromatin contains long single-stranded regions greater than 1 kilobase in length (Kouzine et al., 2017; Bieberstein et al., 2012; Zhou and Paull, 2015). The amount of single-stranded DNA in the genome is estimated to vary from approximately 0.2% to 2.5% depending on the physiological state of the cell (Zhou and Paull, 2015).

[0475] To measure single-stranded DNA in situ with single-cell resolution, CHeX-seq uses the resolution of light to confine reagent action and chromatin analysis to the nucleus of individual cells. To accomplish this, oligonucleotides ( Fig.13A ) have been designed to anneal randomly to single-stranded genomic DNA and remain inactive until photoactivated. After photoactivation, the oligonucleotides serve as primers for DNA polymerase-mediated complementary DNA synthesis ( Fig.13A ). This is DNA-directed in situ transcription (Eberwine et al., 1992; Tecott et al., 1988). The resolution of primer activation is determined by the diffraction limit of the wavelength of the activating light and the numerical aperture of the lens. To facilitate analysis, CHeX-seq oligonucleotides are engineered to contain sample-specific barcodes: a T7 RNA polymerase promoter site and a degenerate DNA sequence that terminates with a fluorescently labeled photoreversibly capped nucleotide ( Figure 3A and Fig.13A After DNA synthesis, the complementary DNA was removed with 0.1 N NaOH, replicated into double-stranded DNA, and linearly amplified using T7 RNA polymerase (aRNA amplification) (Van Gelder et al., 1990; Eberwine et al., 1992). The aRNA was then reverse transcribed into first-strand and second-strand DNA using custom primers, converted into a sequencing library, and sequenced ( Figure 3A and Fig.19).

[0476] CHeX-seq has been benchmarked against human K562 cells analyzed by ENCODE, which demonstrated the utility of CHeX-seq in dispersed mouse and human primary brain cells. These data highlight the significant correlation between open chromatin states and mRNA expression. These data show DNA strand preferences that indicate protein binding domains in single-stranded chromatin. CHeX-seq also provides evidence for genomic DNA regions that exhibit single-stranded strand types but are not transcribed, which may include DNA repair regions and replication sites in dividing cells (Yu et al., 2017; Vasquez et al., 2001). In addition, human neurons and astrocytes are generally found to have more open chromatin than their mouse counterparts. In addition, CHeX-seq can be used for single immunostained cells in fixed brain tissue sections. When CHeX-seq queries single-stranded DNA, it is possible to examine the openness of mitochondria in single cells, and it was found that the DNA in mitochondria present in neurons in situ is more open than those in dispersed cells, indicating differences in metabolic states. The chromosomal landscape of single-stranded open chromatin can be used to classify cells and predict cell subtypes. CHeX-seq enables the analysis of chromatin structure in fixed, immunostained single cells, opening new avenues for examining the role of neuronal circuits in regulating the chromatin landscape of individual cells in their native tissue context.

[0477] Some embodiments provided herein relate to methods that allow the dynamics of chromatin structure and its correspondence with the cytoplasmic RNA pool to be studied simultaneously in single cells. Methods for assessing the dynamics of higher-order chromatin structure around any single gene or multiple genes in anatomically and spatially defined single cells are also provided. Integration of chromatin structure determination with the cytoplasmic transcriptome of the same cell in response to external (e.g., pharmacological) stimulation will enable quantification of the dynamics of cellular transcriptional responses.

[0478] I. Definitions

[0479] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, preferred methods and materials will be described.

[0480] As used herein, "substantially free" with respect to a particular component is used herein to mean that none of the particular component is intentionally formulated into the composition and / or is present only as an impurity or in trace amounts. Thus, the total amount of the specified component resulting from any unintended impurities of the composition is much less than 0.05%, preferably less than 0.01%. Most preferred is a composition in which the amount of the specified component cannot be detected using standard analytical methods.

[0481] As used in this specification, "a / an" may refer to one or more. As used in the claims, when used in conjunction with the word "comprising", the word "a / an" may refer to one or more than one.

[0482] The use of the term "or" in the claims is intended to mean "and / or" unless explicitly indicated to refer to only alternatives or the alternatives are mutually exclusive, although the present disclosure supports definitions and "and / or" that refer only to alternatives. As used herein, "another" may refer to at least a second or more.

[0483] As used herein, "about" refers to a measurable value, such as an amount, duration, etc., and is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are suitable for performing the disclosed methods.

[0484] "Amplification" refers to any means by which a polynucleotide sequence is copied and thereby amplified into a large number of polynucleotide molecules, for example, by methods such as reverse transcription, T7 RNA amplification, polymerase chain reaction, and ligase chain reaction.

[0485] "Sense" refers to the nucleic acid sequence of the coding strand of a double-stranded DNA molecule encoding a protein, or a sequence substantially homologous to the coding strand. As defined herein, the sense sequence is complementary to the sequence of the expressed RNA molecule encoding the protein. The sense sequence need not be complementary only to the coding portion of the expressed RNA molecule. The sense sequence includes a regulatory sequence specified on the coding strand of a DNA molecule encoding a protein, which controls the expression of the coding sequence.

[0486] "Binding" is used herein to mean that a first moiety interacts with a second moiety.

[0487] As used herein, the term "biological sample" refers to a sample obtained from a unicellular or multicellular organism that can be used to assess the expression level of a nucleic acid, the chromatin state, or both. Such samples include, but are not limited to, cells, blood samples, tissue samples, neural tissue samples, brain samples, and cerebrospinal fluid samples.

[0488] As used herein, a "pathological sample" is a biological sample from a subject suffering from or suspected of suffering from a disease, illness, or condition. Pathological samples include, but are not limited to, histological tissue sections and / or other biological products, such as tissue culture cells. Pathological samples are often used to diagnose pathology.

[0489] As used herein, "fixed sample" refers to a sample that has been processed so as to preserve the structural organization of cells and tissues in the sample as close to the true state as possible for subsequent examination, such as by light microscopy. Fixation generally inhibits autolysis and bacterial decomposition, and stabilizes the structural organization of cells and tissues to withstand subsequent stages of tissue processing.

[0490] As used herein, "complementarity" refers to the broad concept of subunit sequence complementarity between two nucleic acids, such as two DNA molecules or a DNA molecule and an RNA molecule. When a nucleotide position in the two molecules is occupied by nucleotides that are usually capable of base pairing with each other, the nucleic acids are considered to be complementary to each other at that position. Therefore, when a considerable number (at least 50%) of corresponding positions in each of the nucleic acid molecules are occupied by nucleotides that are usually base paired with each other (e.g., A:T and G:C nucleotide pairs), the two nucleic acids are complementary to each other.

[0491] The "coding region" of a gene comprises nucleotide residues of the coding strand of the gene and nucleotides of the non-coding strand of the gene, which are respectively homologous or complementary to the coding region of an mRNA molecule produced by transcription of the gene.

[0492] The "coding region" of an mRNA molecule also includes nucleotide residues of the mRNA molecule that match the anticodon region of a transfer RNA molecule during translation of the mRNA molecule, or encode a stop codon. Thus, the coding region may include nucleotide residues that do not exist in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

[0493] As used herein, "degenerate sequence" refers to a sequence in which two or more types of nucleotides are present at one or more of the nucleotide positions in a polynucleotide. In the context of a single polynucleotide, a "degenerate sequence" may be a "random" or "unknown" sequence.

[0494] An "isolated cell" refers to a cell that has been separated from other components and / or cells that naturally accompany the isolated cell in a tissue or organism.

[0495] "Isolated nucleic acid" refers to a nucleic acid (or a segment or fragment thereof) that has been separated from the sequences that flank it in its naturally occurring state, for example, an RNA fragment that has been removed from the sequences that normally adjoin the RNA fragment. The term also applies to nucleic acids that have been substantially purified from other components (e.g., RNA or DNA or proteins) that naturally accompany the nucleic acid in a cell.

[0496] In the context of the present invention, the following abbreviations for common nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to cytidine, and "U" refers to uridine.

[0497] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may contain introns.

[0498] "Polynucleotide" refers to a single strand or parallel and antiparallel strands of a nucleic acid. Thus, a polynucleotide can be a single strand or a double stranded nucleic acid. The term "nucleic acid" generally refers to a large polynucleotide. The term "oligonucleotide" generally refers to a short polynucleotide, generally not greater than about 50 nucleotides. It should be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes RNA sequences in which "U" replaces "T" (i.e., A, U, G, C).

[0499] "Watson / Crick base pairing" and "Watson / Crick complementarity" refer to the pattern in which specific nucleotide pairs and their analogs are held together by hydrogen bonds, such as A pairs with T or U, and G pairs with C. The action of specific base pairing is "hybridization" or "hybridizing." When two or more complementary nucleic acid strands undergo base pairing, a hybrid is formed.

[0500] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction.

[0501] The DNA strand with the same sequence as the mRNA is called the "coding strand"; the sequence on the DNA strand that is 5' of a reference point on the DNA is called the "upstream sequence"; the sequence on the DNA strand that is 3' of a reference point on the DNA is called the "downstream sequence"

[0502] As used herein, a "permeabilizing agent" is a chemical that renders the intracellular structure of a cell accessible to an oligonucleotide or other molecule.

[0503] A "photocleavable moiety" or "photoactivatable moiety" refers to a moiety that is cleaved or activated when the moiety is irradiated with light energy. Light energy that can be used to activate such tags includes, but is not limited to, visible light, ultraviolet (UV) light, infrared (IR) light, and the like. A photocleavable moiety or photoactivatable moiety is "bound" into a nucleic acid when it is attached, bound, integrated, or linked to the nucleic acid. This includes coupling a moiety to the terminus of a nucleic acid, as well as incorporating the moiety into a nucleic acid by including a nucleobase containing such a label.

[0504] "Primer" refers to a polynucleotide that can specifically hybridize with a polynucleotide template and provide a starting point for the synthesis of a complementary polynucleotide. When a polynucleotide primer is placed under conditions of induction synthesis, i.e., in the presence of nucleotides, complementary polynucleotide templates and a polymerizing agent (e.g., DNA polymerase), this synthesis occurs. Primers are typically single-stranded, but may also be double-stranded. Primers are typically deoxyribonucleic acids, but various synthetic and naturally occurring primers may also be used for many applications. Primers are complementary to templates, and the primers are designed to hybridize with the template as a synthesis start site, but do not need to reflect the exact sequence of the template. In this case, the specific hybridization of primers and templates depends on the stringency of hybridization conditions. Primers may be labeled with, for example, a chromogenic portion, a radioactive portion, or a fluorescent portion, and used as a detectable portion.

[0505] "Probe" refers to a polynucleotide that is capable of specific hybridization with a specified sequence of another polynucleotide. The probe specifically hybridizes to a target complementary polynucleotide, but need not reflect the exact complementary sequence of the template. In this case, the specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. The probe can be labeled with, for example, a chromogenic moiety, a radioactive moiety, or a fluorescent moiety and used as a detectable moiety.

[0506] "Genomic DNA" is a DNA strand having a nucleotide sequence homologous to a gene present in the natural host. For example, a chromosome or a chromosome fragment is genomic DNA. In addition, mitochondrial DNA is genomic DNA.

[0507] As used herein, "homologous" refers to the subunit sequence similarity between two polymeric molecules, such as two nucleic acid molecules (e.g., two DNA molecules or two RNA molecules). When the subunit positions of both molecules are occupied by the same monomer subunit, for example, if the position in each of the two DNA molecules is occupied by adenine, the two DNA molecules are completely or 100% homologous at that position. The homology percentage between two sequences is a direct function of the number of matching or homologous positions, for example, if half of the positions in the two compound sequences (e.g., five positions in a polymer of ten subunits in length) are homologous, the two sequences are 50% identical, and if 90% of the positions (e.g., 9 out of 10) are matched or homologous, the two sequences share 90% homology. For example, the DNA sequences 5'ATTGCC3' and 5'TATGGC3' share 50% homology.

[0508] Range: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be construed as an inflexible limitation on the scope of the invention. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within the range. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies no matter how wide the range is.

[0509] When used in the context of a chemical group, "hydrogen" means -H; "hydroxyl" means -OH; "oxo" means =O; "halo" independently means -F, -Cl, -Br or -I; "amino" means -NH2; "hydroxyamino" means -NHOH; "nitro" means -NO2; imino means =NH; "cyano" means -CN; "isocyanate" means -N=C=O; "azido" means -N3; ​​in the monovalent context, "phosphate" means -OP(O)(OH)2 or a deprotonated form thereof; in the divalent context, "phosphate" means -OP(O)(OH)O- or a deprotonated form thereof; "thiol" means -SH; and "thio" means =S; "sulfonyl" means -S(O)2-; and "sulfinyl" means -S(O)-.

[0510] In the context of chemical formulas, the symbol "-" represents a single bond, "=" represents a double bond, and "≡" represents a triple bond. represents an optional bond, which, if present, is a single bond or a double bond. represents a single bond or a double bond. Thus, for example, the structure Including structure As will be appreciated by those skilled in the art, no such ring atom forms part of more than one double bond. When drawn vertically across a bond, it indicates the point of attachment for that group. Note that the point of attachment is usually only indicated in this way for larger groups, in order to help the reader quickly and unambiguously identify the point of attachment. represents a single bond, where the group attached to the thick end of the wedge is "off the page" symbol represents a single bond, where the group attached to the thick end of the wedge "enters the page". The symbol represents a single bond, where the conformation (eg, R or S) or geometry is undefined (eg, E or Z).

[0511] Any undefined valence on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. When the group "R" is described as a "floating group" on a ring system, for example, in the following formula:

[0512]

[0513] Then R may replace any hydrogen atom attached to any of the ring atoms, including depicted, implied or explicitly defined hydrogens, as long as a stable structure results. When the group "R" is described as a "floating group" on a fused ring system, for example in the following formula:

[0514]

[0515] Then unless otherwise stated, R can replace any hydrogen of any ring atom of any ring atom attached to any one of the condensed rings.Substitutable hydrogen includes depicted hydrogen (e.g., hydrogen attached to nitrogen in the above formula), implicit hydrogen (e.g., hydrogen not shown in the above formula but understood to be present), clearly defined hydrogen, and optional hydrogen, whose presence depends on the identity of the ring atom (e.g., when X is equal to -CH-, it is hydrogen attached to the group X), as long as a stable structure is formed. In the depicted example, R can be located on a 5-membered ring or a 6-membered ring of a condensed ring system. In the above formula, the subscript "y" following the group "R" enclosed by brackets represents a numerical variable. Unless otherwise stated, the variable can be 0, 1, 2, or any integer greater than 2, limited only by the maximum number of replaceable hydrogen atoms in the ring or ring system.

[0516] For the following groups and classes, the following bracketed subscripts further define the group / class as follows: "(Cn)" defines the exact number of carbon atoms (n) in the group / class. "(C≤n)" defines the maximum number of carbon atoms (n) that can be in the group / class, where the minimum number is as small as possible for the group in question, e.g., it is understood that the group "alkenyl" (C≤8) " or the category "alkenyl (C≤8) The minimum number of carbon atoms in an alkoxy group is 2. (C≤10) ” refers to those alkoxy groups having 1 to 10 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range derivable therein (e.g., 3 to 10 carbon atoms)). (Cn-n′) defines the minimum number (n) and maximum number (n′) of carbon atoms in the group. Similarly, “alkyl” (C2-10) ” refers to those alkyl groups having from 2 to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range derivable therein (e.g., 3 to 10 carbon atoms)).

[0517] As used herein, the term "saturated" refers to compounds or groups that, unless noted below, are modified so as to be free of carbon-carbon double bonds and carbon-carbon triple bonds. The term does not exclude carbon-heteroatom multiple bonds, such as carbon-oxygen double bonds or carbon-nitrogen double bonds. Furthermore, it does not exclude carbon-carbon double bonds that may occur as part of keto-enol tautomerism or imine / enamine tautomerism.

[0518] The term "aliphatic" when used without the "substituted" modifier means that the compound / group so modified is acyclic or cyclic, but is a non-aromatic hydrocarbon compound or group. In an aliphatic compound / group, the carbon atoms can be linked together in a straight chain, branched chain or non-aromatic ring (alicyclic). Aliphatic compounds / groups can be saturated, i.e. linked by single bonds (alkanes / alkyls), unsaturated, having one or more double bonds (alkenes / alkenyls) or having one or more triple bonds (alkynes / alkynyls). When the term "aliphatic" is used without the "substituted" modifier, only carbon and hydrogen atoms are present. When this term is used with the "substituted" modifier, one or more hydrogen atoms have independently been replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -OC(O)CH3, or -S(O)2NH2.

[0519] The term "alkyl" when used without the "substituted" modifier refers to a monovalent saturated aliphatic group having a carbon atom as the point of attachment, a straight chain or branched, cyclic, cyclic or acyclic structure, and no atoms other than carbon and hydrogen. Thus, as used herein, cycloalkyl is a subset of alkyl. The groups -CH3(Me), -CH2CH3(Et), -CH2CH2CH3(n-Pr or propyl), -CH(CH3)2(i-Pr, i Pr or isopropyl), -CH(CH2)2(cyclopropyl), -CH2CH2CH2CH3(n-Bu), -CH(CH3)CH2CH3(sec-butyl), -CH2CH(CH3)2(isobutyl), -C(CH3)3(tert-butyl, t-butyl, t-Bu or t Bu), -CH2C(CH3)3 (neopentyl), cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl are non-limiting examples of alkyl groups. The term "alkanediyl" when used without the "substituted" modifier refers to a divalent saturated aliphatic radical having one or two saturated carbon atoms as points of attachment, a straight chain or branched, cyclic, cyclic or acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The radicals -CH2- (methylene), -CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2CH2- and is a non-limiting example of an alkanediyl group. The term "alkylene" when used without the "substituted" modifier refers to a divalent group =CRR', wherein R and R' are independently hydrogen, alkyl, or R and R' together represent an alkanediyl group having at least two carbon atoms. Non-limiting examples of alkylene groups include: =CH2, =CH(CH2CH3), and =C(CH3)2. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -OC(O)CH3, or -S(O)2NH2. The following groups are non-limiting examples of substituted alkyl groups: -CH2OH, -CH2Cl, -CF3, -CH2CN, -CH2C(O)OH, -CH2C(O)OCH3, -CH2C(O)NH2, -CH2C(O)CH3, -CH2OCH3, -CH2OC(O)CH3, -CH2NH2, -CH2N(CH3)2, and -CH2CH2Cl. The term "haloalkyl" is a subset of substituted alkyl groups in which one or more hydrogen atoms have been replaced by a halogen group and there are no other atoms except carbon, hydrogen and halogen. The group -CH2Cl is a non-limiting example of a haloalkyl group. "Alkane" refers to the compound HR in which R is an alkyl group. The term "fluoroalkyl" is a subset of substituted alkyl groups in which one or more hydrogen atoms have been replaced by a fluorine group and there are no other atoms except carbon, hydrogen and fluorine. The groups -CH2F, -CF3 and -CH2CF3 are non-limiting examples of fluoroalkyl groups. "Alkane" refers to the compound HR wherein R is an alkyl group.

[0520] The term "alkenyl" when used without the "substituted" modifier refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a straight chain or branched, cyclic, cyclic or acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples of alkenyl groups include: -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCH3, and -CH=CH-C6H5. The term "alkenediyl" when used without the "substituted" modifier refers to a divalent unsaturated aliphatic group having two carbon atoms as the point of attachment, a straight chain or branched, cyclic, cyclic or acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH3)CH2-, -CH=CHCH2-, and are non-limiting examples of olefinic diyls. When these terms are used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -OC(O)CH3, or -S(O)2NH2. The groups -CH=CHF, -CH=CHCl, and -CH=CHBr are non-limiting examples of substituted olefinic diyls. "Olefin" refers to the compound HR in which R is an olefinic group.

[0521] The term "alkynyl" when used without the "substituted" modifier refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a straight chain or branched, cyclic, cyclic or acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not exclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH3, and -CH2C≡CCH3 are non-limiting examples of alkynyl groups. When alkynyl is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -OC(O)CH3, or -S(O)2NH2. "Alkyne" refers to the compound HR in which R is alkynyl.

[0522] The term "aryl" when used without a "substituted" modifier refers to a monovalent unsaturated aromatic group having an aromatic carbon atom as a point of attachment, the carbon atom forming a part of one or more six-membered aromatic ring structures, wherein the ring atoms are all carbon, and wherein the group is not composed of other atoms except carbon and hydrogen. If there are more than one ring, these rings can be fused or non-fused. As used herein, the term does not exclude the presence of one or more alkyl groups (carbon number restrictions allow) attached to the first aromatic ring or any additional aromatic rings present. Non-limiting examples of aryl include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C6H4CH2CH3 (ethylphenyl), naphthyl, and a monovalent group derived from biphenyl. The term "arenediyl" when used without the "substituted" modifier refers to a divalent aromatic group having two aromatic carbon atoms as points of attachment, the carbon atoms forming part of one or more six-membered aromatic ring structures, wherein the ring atoms are all carbon, and wherein the monovalent groups are not composed of atoms other than carbon and hydrogen. As used herein, the term does not exclude the presence of one or more alkyl groups (carbon number limitations permitting) attached to the first aromatic ring or any additional aromatic rings present. If more than one ring is present, the rings may be fused or non-fused. Non-limiting examples of arenediyl include:

[0523]

[0524] When these terms are used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -OC(O)CH3, or -S(O)2NH2. "Aromatic" refers to the compound HR in which R is an aryl group.

[0525] The term "aralkyl" refers to monovalent groups-alkanediyl-aryl when used without "substituted" modifiers, wherein the terms alkanediyl and aryl are used in a manner consistent with the definition provided above. The non-limiting examples of aralkyl are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When the term is used together with the "substituted" modifier, one or more hydrogen atoms from alkanediyl and / or aryl have been independently replaced by-OH ,-F ,-Cl ,-Br ,-I ,-NH ,-NO ,-CO H ,-CO CH ,-CN ,-SH ,-OCH ,-OCH CH ,-C (O) CH ,-NHCH ,-NHCH CH ,-N (CH ) ,-C (O) NH ,-OC (O) CH , or-S (O) NH . The non-limiting examples of the substituted aralkyl are: (3-chlorophenyl)-methyl and 2-chloro-2-phenyl-second-1-yl.

[0526] The term "heteroaryl" when used without the "substituted" modifier refers to a monovalent aromatic group having an aromatic carbon atom or nitrogen atom as a point of attachment, the carbon atom or nitrogen atom forming part of one or more aromatic ring structures, wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the heteroaryl group is not composed of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. As used herein, the term does not exclude the presence of one or more alkyl, aryl and / or aralkyl groups (carbon number limitations permitting) attached to an aromatic ring or aromatic ring system. If more than one ring is present, the rings may be fused or non-fused. Non-limiting examples of heteroaryl include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridyl, oxazolyl, phenylpyridyl, pyridyl, pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolinyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl and triazolyl. The term "N-heteroaryl" refers to a heteroaryl with a nitrogen atom as a point of attachment. The term "heteroaryldiyl" when used without a "substituted" modifier refers to a divalent aromatic group having two aromatic carbon atoms, two aromatic nitrogen atoms or one aromatic carbon atom and one aromatic nitrogen atom as two points of attachment, the atoms forming part of one or more aromatic ring structures, wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the divalent group is not composed of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. As used herein, the term does not exclude the presence of one or more alkyl, aryl and / or aralkyl groups (carbon number limitations permitting) attached to an aromatic ring or aromatic ring system. If more than one ring is present, the rings may be fused or non-fused. Non-limiting examples of heteroaryl diyls include:

[0527]

[0528] When these terms are used with the "substituted" modifier, one or more hydrogen atoms have independently been replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -OC(O)CH3, or -S(O)2NH2.

[0529] The term "heterocycloalkyl" when used without the "substituted" modifier refers to a monovalent non-aromatic group having a carbon atom or a nitrogen atom as a point of attachment, the carbon atom or nitrogen atom forming part of one or more non-aromatic ring structures, wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the heterocycloalkyl group is not composed of atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. As used herein, the term does not exclude the presence of one or more alkyl groups (carbon number restrictions permitting) attached to a ring or ring system. As used herein, the term does not exclude the presence of one or more double bonds in a ring or ring system, provided that the resulting group remains non-aromatic. If there are more than one ring, the rings may be fused or non-fused. Non-limiting examples of heterocycloalkyl include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term "N-heterocycloalkyl" refers to a heterocycloalkyl group with a nitrogen atom as the point of attachment. When the term "heterocycloalkyl" is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -OC(O)CH3, -S(O)2NH2, or -C(O)OC(CH3)3 (tert-butyloxycarbonyl, BOC).

[0530] The term "acyl" when used without the "substituted" modifier refers to the group -C(O)R, wherein R is hydrogen, alkyl, aryl, aralkyl or heteroaryl, as those terms are defined above. The groups -CHO, -C(O)CH3(acetyl, Ac), -C(O)CH2CH3, -C(O)CH2CH2CH3, -C(O)CH(CH3)2, -C(O)CH(CH2)2, -C(O)C6H5, -C(O)C6H4CH3, -C(O)CH2C6H5, -C(O)(imidazolyl) are non-limiting examples of acyl. "Thioacyl" is defined in a similar manner, except that the oxygen atom of the group -C(O)R has been replaced by a sulfur atom, -C(S)R. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms (including those directly attached to the carbonyl or thiocarbonyl group, if any) have been independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -OC(O)CH, or -S(O)NH. The groups -C(O)CHCF, -COH (carboxyl), -COCH (methylcarboxyl), -COCHCH, -C(O)NH (carbamoyl), and -CON(CH) are non-limiting examples of substituted acyl groups.

[0531] The term "alkoxy" when used without the "substituted" modifier refers to a group -OR, wherein R is an alkyl group, as defined above. Non-limiting examples of alkoxy groups include: -OCH3 (methoxy), -OCH2CH3 (ethoxy), -OCH2CH2CH3, -OCH(CH3)2 (isopropoxy), -O(CH3)3 (tert-butoxy), -OCH(CH2)2, -O-cyclopentyl and -O-cyclohexyl. The terms "alkenyloxy", "alkynyloxy", "aryloxy", "aralkyloxy", "heteroaryloxy", "heterocycloalkyloxy" and "acyloxy" when used without the "substituted" modifier refer to a group defined as -OR, wherein R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl and acyl, respectively. The term "alkoxydiyl" refers to a divalent group -O-alkanediyl-, -O-alkanediyl-O- or -alkanediyl-O-alkanediyl-. The terms "alkylthio" and "acylthio" when used without the "substituted" modifier refer to the group -SR, wherein R is alkyl and acyl, respectively. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -OC(O)CH, or -S(O)NH. The term "alcohol" corresponds to an alkane as defined above, wherein at least one of the hydrogen atoms is replaced by a hydroxyl group.

[0532] The term "alkylamino" when used without the "substituted" modifier refers to the group -NHR, where R is an alkyl group, as that term is defined above. Non-limiting examples of alkylamino groups include: -NHCH3 and -NHCH2CH3. The term "dialkylamino" when used without the "substituted" modifier refers to the group -NRR', where R and R' can be the same or different alkyl groups, or R and R' can together represent an alkanediyl group. Non-limiting examples of dialkylamino groups include: -N(CH3)2, -N(CH3)(CH2CH3), and N--pyrrolidinyl. The terms "alkoxyamino", "alkenylamino", "alkynylamino", "arylamino", "aralkylamino", "heteroarylamino", "heterocycloalkylamino", and "alkylsulfonylamino" when used without the "substituted" modifier refer to the group defined as -NHR, where R is alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and alkylsulfonyl, respectively. A non-limiting example of an arylamino group is -NHC6H5. The term "acylamino" (acylamino), when used without a "substituted" modifier, refers to a group -NHR, where R is acyl, as that term is defined above. A non-limiting example of an amide group is -NHC(O)CH3. The term "alkylimino," when used without a "substituted" modifier, refers to a divalent group =NR, where R is alkyl, as that term is defined above. The term "alkylaminodiyl" refers to a divalent group -NH-alkanediyl-, -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -OC(O)CH, or -S(O)NH. The groups -NHC(O)OCH and -NHC(O)NHCH are non-limiting examples of substituted amide groups.

[0533] The term "alkylphosphate" when used without the "substituted" modifier refers to the group -OP(O)(OH)(OR), in which R is an alkyl group, as that term is defined above. Non-limiting examples of alkylphosphate groups include: -OP(O)(OH)(OMe) and -OP(O)(OH)(OEt). The term "dialkylphosphate" when used without the "substituted" modifier refers to the group -OP(O)(OR)(OR'), in which R and R' can be the same or different alkyl groups, or R and R' can together represent an alkanediyl group. Non-limiting examples of dialkylphosphate groups include: -OP(O)(OMe)2, -OP(O)(OEt)(OMe), and -OP(O)(OEt)2. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have independently been replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -OC(O)CH3, or -S(O)2NH2.

[0534] The terms "alkylsulfonyl" and "alkylsulfinyl" when used without the "substituted" modifier refer to the groups -S(O)2R and -S(O)R, respectively, where R is alkyl, as that term is defined above. The terms "alkenylsulfonyl," "alkynylsulfonyl," "arylsulfonyl," "aralkylsulfonyl," "heteroarylsulfonyl," and "heterocycloalkylsulfonyl" are defined in a similar manner. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -OC(O)CH3, or -S(O)2NH2.

[0535] As used herein, "chiral auxiliary" refers to a removable chiral group that is capable of affecting the stereoselectivity of a reaction. Those skilled in the art are familiar with such compounds, and many are commercially available.

[0536] "Isomers" of a first compound are individual compounds wherein each molecule contains the same constituent atoms as the first compound, but in a different three-dimensional configuration of those atoms.

[0537] The term "hydrate" when used as a modifier of a compound refers to a compound that has less than one (e.g., hemihydrate), one (e.g., monohydrate), or more than one (e.g., dihydrate) water molecule associated with each molecule of the compound, such as in a solid form of the compound.

[0538] "Stereoisomers" or "optical isomers" are isomers of a given compound in which the same atoms are bonded to the same other atoms, but the three-dimensional configuration of these atoms is different. "Enantiomers" are stereoisomers of a given compound that are mirror images of each other, such as left and right hands. "Diastereomers" are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain chiral centers, also called stereocenters or stereogenic centers, which are any points in a molecule that bear groups, although not necessarily atoms, such that the interchange of any two groups will produce stereoisomers. In organic compounds, chiral centers are usually carbon, phosphorus, or sulfur atoms, although other atoms may also be stereocenters in organic and inorganic compounds. A molecule can have multiple stereocenters, giving it multiple stereoisomers. In compounds where stereoisomerism is due to tetrahedral stereocenters (e.g., tetrahedral carbon), it is assumed that the total number of possible stereoisomers will not exceed 2n, where n is the number of tetrahedral stereocenters. Molecules with symmetry tend to have less than the maximum possible number of stereoisomers. A 50:50 mixture of enantiomers is called a racemic mixture. Alternatively, the enantiomeric mixture can be enantiomeric enriched so that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and / or diastereomers can be split or separated using techniques known in the art. It is envisioned that for any stereocenter or chiral axis for which stereochemistry has not yet been defined, the stereocenter or chiral axis can exist in its R form, S form, or as a mixture of R and S forms, including racemic and non-racemic mixtures. As used herein, the phrase "substantially free of other stereoisomers" means that the composition contains ≤15%, more preferably ≤10%, even more preferably ≤5%, or most preferably ≤1% of additional stereoisomers.

[0539] II. Compositions and methods for detecting open genomic DNA (CHeX-seq)

[0540] Chromatin accessibility underlies the ability of cells to respond transcriptionally to their local microenvironment and stimuli. This is important for normal cellular function as well as regulatory changes in function. Knowledge of these subcellular sites of transcriptional regulation is essential to understanding the ability of cells to respond to stimuli as well as the actual responses.

[0541] Transcriptional analysis of single cells has shown that a significant degree of cell-to-cell heterogeneity is affected by the cell's microenvironment. These changes in transcriptional state depend in part on the open chromatin state of nuclear genomic DNA or the open conformation of mitochondrial genomic DNA. Although there are single-cell methods for analyzing chromatin structure, they require the chromatin to be isolated from the cell, at which point the nearest neighboring chromosome interactions are lost. For example, ATACseq has been used to assess open chromatin in multiple single cells (population studies at the single-cell level, not just single cells), but it is not very sensitive and only assesses about 3% of open chromatin sites in any particular cell. In addition, ATACseq requires the chromatin to be isolated from the cell of interest.

[0542] To overcome these problems of ATACseq and other chromosome conformation capture methods, this paper provides high-resolution single-cell chromatin analysis methods that reflect the functional state of cells. These methods, called CHeX-seq (Chromatin Exposure), do not require the isolation of genomic DNA from cells and can be used to identify open genomic DNA regions in single fixed cells.

[0543] High-resolution CHeX-seq uses the resolution of light to confine chromatin analysis to the nucleus of individual cells. To achieve this, CHeX-seq oligonucleotides have been engineered to contain an index barcode, an amplification segment, and a reversible terminator nucleotide ( Figure 1 ). These oligonucleotides can anneal to single-stranded genomic DNA in a random or targeted manner through their hybridization segments, but remain inactive until triggered. The reversible termination nucleotides labeled with photoactivated reporters that are activated in situ in selected cells by lasers allow the CHeX-seq oligonucleotides to be used as primers. The primers are used to initiate DNA replication of single-stranded DNA in situ in these specific cells, which is then amplified and sequenced.

[0544] In order to provide for random annealing, oligonucleotide comprises short degenerate sequence, and described short degenerate sequence can anneal anywhere they can hybridize with genomic DNA.This provides more oligonucleotides that can be combined in degenerate mixture, thereby effectively increases oligonucleotide concentration in the process of annealing time (for example, 45 seconds).In some embodiments, annealing time can be about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55 seconds, about 60 seconds, about 65 seconds, about 70 seconds, about 75 seconds, about 80 seconds, about 85 seconds, about 90 seconds, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes or about 30 minutes.The process of annealing can be monitored using fluorescence microscope, to detect the fluorescent signal of fluorescent labeling in nucleus.

[0545] Hybridization between the degenerate sequence in the oligonucleotide and the genomic DNA may not be strict.For example, there may be one or more, two or more, 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 mispairings between the degenerate sequence in the oligonucleotide and the genomic DNA region that hybridizes with it.The mispairing quantity that can be tolerated while still allowing hybridization with any given genomic DNA sequence depends on the length of the degenerate sequence, the hybridization thermodynamics indicated by the sequence of the degenerate sequence and the annealing temperature.The thermodynamics of hybridization events can be estimated using the method described in SantaLucia and Hicks (2004).Mispairing can be one or more continuous segments, or mispairing can be spaced apart in the whole hybridization region with any configuration.For example, the hybridization region can include a row with two, three, four, five, six, seven or eight mispairings, and these mispairings produce bubbles in the hybridization region.The hybridization region can include both the bubble in the first part and the single mispairing in the second part.The hybridization region can include both the first bubble in the first part and the second bubble in the second part.

[0546] The length of the degenerate sequence can vary. For example, the resulting sequence in any given CHeX-seq oligonucleotide can be at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. A CHeX-seq oligonucleotide population can be composed of oligonucleotides that all have degenerate sequences of the same length. Alternatively, the CHeX-seq oligonucleotide population can contain oligonucleotides having degenerate sequences of varying lengths.

[0547] In addition to providing for hybridization with genomic DNA, the degenerate sequence will be retained in the subsequently amplified polynucleotide pool, which is analyzed using sequencing (e.g., next generation sequencing). Thus, the degenerate sequence associated with each detected hybridization event can be used as a molecular barcode or unique molecular identifier (UMI) to allow analysis of whether only one locus of a gene is in an open chromatin state, or whether both loci of the gene are in an open chromatin state.

[0548] In order to provide for targeted annealing, oligonucleotide comprises short known sequence to replace short degenerate sequence.Short known sequence can be designed to anneal with any position of genomic DNA that needs to analyze chromatin whether it is open or closed state.For example, one group of oligonucleotide can be designed to comprise short known sequence limited for one or more specific genes.For example, one group of oligonucleotide can be designed to comprise short known sequence, and described short known sequence can hybridize with the genome region near all known SNPs or with the open genomic DNA region relevant to schizophrenia.This oligonucleotide group can be used in diagnostic method.When designing one group of targeted oligonucleotide, it may be desirable to select short known sequence with certain G / C content and length so that each oligonucleotide will hybridize under similar annealing temperature.

[0549] CHeX-seq oligonucleotides can have a reporter-tagged reversible stop nucleotide at their 3' end. Figure 3A , Figure 3B and Figure 4Two exemplary embodiments of methods for enzymatically synthesizing oligonucleotides containing reporter-tagged reversible terminator nucleotides are shown. These methods include annealing an oligonucleotide to its reverse complement sequence, wherein the reverse complement sequence contains a poly A tail on its 5' end. Once annealed, the duplex is incubated with a fluorescently labeled photoactivatable terminator deoxyuridine analog that is incorporated into the oligonucleotide by a DNA polymerase. The synthesized oligonucleotide can then be denatured from its reverse complement sequence and purified. The method can be performed with an intact oligonucleotide and its intact reverse complement sequence ( Figure 3A Alternatively, the method can be performed with a segment comprising a degenerate sequence, followed by joining the double-stranded degenerate sequence with a segment comprising an amplification segment, a spacer, and a barcode ( Figure 3B ). This method allows the generation of many different populations, each containing a unique barcode, without having to synthesize a double-stranded degenerate sequence for each population. To this end, only a segment containing an amplification segment, a spacer, and a barcode is generated for each population, and each unique barcode can then be linked to a double-stranded degenerate sequence to generate each uniquely barcoded degenerate population. Another alternative method uses a reverse complement sequence with a 6-carbon linker instead of a degenerate sequence ( Figure 4 ). In this method, the oligonucleotide has a TG clamp at its 3′ end to promote hybridization with the reverse complementary strand, thereby allowing the addition of a photoactivatable terminator nucleotide.

[0550] In some embodiments, the reporter-labeled reversible terminator nucleotide compound comprises a nucleotide having a photocleavable group labeled with a reporter group, such as a fluorescent dye group, a colorimetric dye group, a radioactive label, or a group that affects a signal by chemiluminescence or bioluminescence. As used herein, the term "reporter" or "label" refers to a chemical moiety that can directly or indirectly produce a detectable signal. The terminator nucleotide comprises a removable protective group that is designed to terminate DNA synthesis. Examples of such nucleotide compounds include those disclosed in PCT Publication Nos. WO 2003 / 006625, WO 2005 / 084367, WO 2008 / 070749, WO 2009 / 152353, and WO 2013 / 040257, each of which is incorporated herein by reference in its entirety. Specific examples of such nucleotide compounds include Figure 5Examples of indirectly detectable reporters include small tags, such as biotin, haptens (e.g., digoxigenin), or magnetic particles, which can be detected by binding to another protein or antibody so that the reporter can be detected and visualized under a microscope. Examples of directly detectable reporters include fluorescent dye groups. Examples of fluorescent dye groups include xanthene derivative dyes (e.g., fluorescein and its derivatives, fluorescein isothiocyanate [FITC], carboxyfluorescein succinimidyl ester [CFSE], carboxyfluorescein diacetate succinimidyl ester [CFDA-SE], eosin Y, eosin B, rhodamine B, rhodamine 6G, rhodamine 123, rhodamine red-X [RRX], carboxytetramethylrhodamine [TAMRA], tetramethylrhodamine [TMR], isothiocyanate derivatives of rhodamine [TRITC], sulforhodamine 101, sulfonyl chloride derivatives of sulforhodamine 101 [Texas Red], Oregon Green), BODIPY derivative dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY 581 / 591, BODIPY TR, BODIPY 630 / 650, BODIPY 650 / 665), coumarin-derivative dyes (e.g., aminomethylcoumarin [AMCA]), allophycocyanin [APC], pyrene-derivative dyes (e.g., Cascade Blue), 4′,6-diamidino-2-phenylindole [DAPI], DyLight dyes (e.g., DyLight TM 350、DyLight TM 405. DyLight TM 488、DyLight TM 550、DyLight TM 594、DyLight TM 633、DyLight TM 650、DyLight TM 680、DyLight TM 755. DyLight TM 800), phycoerythrin [PE], PI, peridinin-chlorophyll-protein [PerCP], cyanine derivative dyes (e.g., Indole dicarbocyanine Cyanine Indocarbocyanine Oxocarbocyanine, thiocarbocyanine, merocyanine, phthalocyanine), anthracene derivative dyes (e.g., Draq-5, Draq-7, CyTRAK Orange, IRIS 2, IRIS3, IRIS 3.5, IRIS 5, IRIS 5.5, IRIS 7G), eFluor dyes (e.g., 450, 615, 660, 710, 610, 710, 780), FluoProbes dyes (FluoProbes 390, FluoProbes 488, FluoProbes 532, FluoProbes547H, FluoProbes 594, FluoProbes 647H, FluoProbes 682, FluoProbes 752, FluoProbes782), GFP, IRDye 800, Pacific Blue, Pacific Green, Pacific Orange, pyrene, phycobiliprotein, Dyes (e.g. 570, 670, 705), SNAFL, sulfocyanine derivative dyes (e.g., sulfo-Cy3, sulfo-Cy5, sulfo-Cy7), Tokyo Green, Dyes (e.g. Squaric acid dyes (e.g., Seta TM dye, SeTau dye, Square dye), or a combination thereof.

[0551] The first process in performing CHeX-seq is to obtain and fix the sample to be measured, which can be a cell line, a primary cell culture or a tissue section. The sample can contain any type of cell, such as a eukaryotic cell or a prokaryotic cell. When the cell is a eukaryotic cell, the cell is preferably a mammalian cell, including but not limited to human, non-human primate, mouse, rabbit, rat, goat, guinea pig, horse cell, etc. Non-mammalian eukaryotic cells include yeast cells, plant cells, insect cells, protozoan cells and fungal cells, including filamentous and non-filamentous fungi. When the cell is a prokaryotic cell, the cell is a bacterial cell. The cell can be a differentiated cell and / or a non-dividing cell. The cell can also be a progenitor cell or a stem cell. Preferably, the cell is a tissue-specific cell, more preferably a mammalian tissue-specific cell, and more preferably a human tissue-specific cell. Non-limiting examples of cells suitable as receptor cells include epithelial cells, neurons, fibroblasts, embryonic fibroblasts, keratinocytes, adult stem cells, embryonic stem cells and cardiomyocytes. The method provided herein can be performed on cells containing cell protrusions. Such cell processes include, but are not limited to, dendrites, axons, microvilli, cilia, stereocilia, processes, astrocyte processes, etc. Any tissue sample from a subject can be used in the method of the present invention. Examples of tissues that can be used include, but are not limited to, breast, prostate, ovary, colon, lung, endometrium, stomach, salivary gland, and pancreas. Tissue samples can be obtained by various procedures, including, but not limited to, surgical excision, aspiration, or biopsy. Tissue can be embedded in paraffin or frozen. Tissue samples can be stained to distinguish between cell types within the sample, such as distinguishing neurons and glia in brain samples.

[0552] In some embodiments, the sample can be exposed to a permeabilizing agent to allow the oligonucleotide to enter the cell. Exemplary permeabilizing agents include, but are not limited to, Triton X-100, Tween-20, saponin, SDS, NP40, streptolysin O, proteinase K, pronase, and triethanolamine, as well as organic solvents such as methanol and acetone.

[0553] A technical consideration in performing CHeX-seq is to allow CHeX-seq oligonucleotides to enter the nucleus and hybridize them with genomic DNA. The accessibility of nuclear DNA is determined in part by the fixation conditions. For example, using a low percentage of fixative (e.g., 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, or 8%) for 30 seconds, 45 seconds, 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, 6.5 minutes, 7 minutes, 7.5 minutes, 8 minutes, 8.5 minutes, 9 minutes, 9.5 minutes, or 10 minutes can provide detection of open genomic DNA for about 80% of the expressed RNA, which is about 8 times better than reported for ATACseq coverage. The fixative can be formalin, glutaraldehyde, xylene, precipitating fixatives (such as methanol or ethanol), or chemical and photoreversible crosslinkers.

[0554] Once the CHeX-seq oligonucleotide is present in the nucleus, the oligonucleotide is given time to anneal to the region of open genomic DNA. In some embodiments, the annealing time can be about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55 seconds, about 60 seconds, about 65 seconds, about 70 seconds, about 75 seconds, about 80 seconds, about 85 seconds, or about 90 seconds. In some embodiments, the annealing temperature can be about 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C. The progress of annealing can be monitored using a fluorescence microscope to detect the fluorescent signal of the fluorescent marker in the nucleus.

[0555] The hybridized oligonucleotides are then imaged using their reporter tags. In some embodiments, the reporter-tagged reversibly terminated oligonucleotides are reversibly terminated due to the presence of a site that can be photocleaved, thereby producing an extendable 3' hydroxyl. In some embodiments, the photocleavable moiety may include a 2-nitrobenzyl or substituted 2-nitrobenzyl, which can be photochemically cleaved effectively, for example, with ultraviolet light. See U.S. Patent Application Publication 2010 / 0041041, the full text of which is incorporated herein by reference. It is generally understood that a wavelength of >300nm is used to minimize damage to DNA and proteins (Corrie, 2005), wherein several specific exemplary wavelengths other than 365nm are 340nm and 355nm (Seo, 2005). Therefore, the terms "photocleaving" or "photocleave" as used herein generally refer to the action of exposing a sample to light of a wavelength >300 nm (e.g., 365 nm, 370 nm, 375 nm, 380 nm, 385 nm, 390 nm, 395 nm, 400 nm, 405 nm, 410 nm, or 415 nm) to achieve cleavage of a photocleavable bond.

[0556] In some aspects, the photoactivatable terminating nucleotide has the structure of the formula:

[0557]

[0558] in:

[0559] R1 is monophosphate, diphosphate, triphosphate, α-thiotriphosphate, thiophosphate, or polyphosphate;

[0560] R2 is hydrogen or hydroxyl;

[0561] R3 is an alkyl group (C≤8) or substituted alkyl (C≤8) ;

[0562] R4 is

[0563] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0564] alkyl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0565] R5, R6 and R7 are each independently:

[0566] hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol;

[0567] alkyl (C≤6) , alkenyl (C≤6) , alkynyl (C≤6) , aromatic (C≤6) , arylalkyl (C≤8) , heteroaryl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups;

[0568] A set of the following formulas:

[0569]

[0570] in

[0571] X is

[0572] -O-, -S-, or -NH-; or

[0573] Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , or a substituted form of any of these groups;

[0574] Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ;

[0575] n is an integer from 0 to 6; and

[0576] m is an integer from 0 to 6; or

[0577] - linker-reporter;

[0578] or a salt, tautomer or optical isomer thereof.

[0579] In some embodiments, the photoactivatable terminator nucleotide is further defined as a compound of Formula I, II, III, IV, V, VI, or VII. In some embodiments, R1 is hydroxyl, monophosphate, diphosphate, triphosphate, α-thiotriphosphate, or polyphosphate. In some embodiments, R2 is hydrogen, hydroxyl. In some embodiments, R3 is alkyl. (C≤8) , such as alkyl (C3-4), including isopropyl or tert-butyl. In some embodiments, R4 is hydrogen, nitro. In some embodiments, R5 is hydrogen, iodine or alkoxy. (C≤6) , including, for example, methoxy. In some embodiments, R5 is a group of the formula:

[0580]

[0581] in

[0582] X is

[0583] -O-, -S-, or -NH-; or

[0584] Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , aromatic diyl (C≤12) , heteroaryldiyl (C≤12) , or a substituted form of any of these groups; and

[0585] n is an integer between 0-6.

[0586] In some embodiments, X is an acetylenic diyl (C2-8) , for example -C≡C-. In some embodiments, n is zero. In some embodiments, R5 is a group of the formula:

[0587]

[0588] in

[0589] X is

[0590] -O-, -S-, or -NH-; or

[0591] Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , aromatic diyl (C≤12) , heteroaryldiyl (C≤12) , or a substituted form of any of these groups;

[0592] Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ;

[0593] n is an integer from 0 to 6; and

[0594] m is an integer from 0 to 6.

[0595] In some embodiments, X is an acetylenic diyl (C2-8), e.g., -C≡C-. In some embodiments, Y is -CH2-. In some embodiments, n is zero. In some embodiments, m is zero. In some embodiments, R5 is -linker-reporter. In some embodiments, the linker is:

[0596]

[0597] in

[0598] X is

[0599] -O-, -S-, or -NH-; or

[0600] Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , aromatic diyl (C≤12) , heteroaryldiyl (C≤12) , or a substituted form of any of these groups; and

[0601] n is an integer between 0-6.

[0602] In some embodiments, X is an acetylenic diyl (C2-8) , e.g. -C≡C-. In some embodiments, n is zero. In some embodiments, the linker is:

[0603]

[0604] in

[0605] X is

[0606] -O-, -S-, or -NH-; or

[0607] Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , aromatic diyl (C≤12) , heteroaryldiyl (C≤12) , or a substituted form of any of these groups;

[0608] Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ;

[0609] n is an integer from 0 to 6; and

[0610] m is an integer from 0 to 6.

[0611] In some embodiments, X is an acetylenic diyl (C2-8), e.g., -C≡C-. In some embodiments, Y is -CH2-. In some embodiments, n is zero. In some embodiments, m is zero. In some embodiments, the reporter is based on a dye, wherein the dye is a zanthene, fluorescein, rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine, phycobiliprotein, or a squaric acid dye.

[0612] In some embodiments, R6 is hydrogen. In some embodiments, the star-shaped carbon atom is in the S configuration. In some embodiments, the star-shaped carbon atom is in the R configuration. In some embodiments, R7 is methoxy.

[0613] In some embodiments, the photoactivatable terminating nucleotide is further defined as:

[0614]

[0615] or a salt and / or protonated form of any of these formulae.

[0616] In some embodiments, the photoactivatable terminating nucleotide is further defined as:

[0617]

[0618] wherein R is =0 or =S, or a salt and / or protonated form of any of these formulae.

[0619] In some embodiments, the photoactivatable terminating nucleotide is further defined as:

[0620]

[0621]

[0622] wherein R is =0 or =S, or a salt and / or protonated form of any of these formulae.

[0623] In some embodiments, the photoactivatable terminating nucleotide is further defined as:

[0624]

[0625]

[0626]

[0627]

[0628] or a salt and / or protonated form of any of these formulae.

[0629] In some embodiments, after imaging, a reporter-tagged photoactivatable terminator oligonucleotide is photoactivated in a specific region or the entire cell nucleus using, for example, a 405 nm laser. The resolution of photoactivation (which is the removal of the terminator moiety on the nucleotide compound) is determined by the diffraction limit of the wavelength of the activating light and the numerical aperture of the lens. For example, when multiphoton activation is used, the region to be photoactivated can be a 10 nm sphere (see Figure 6 ). In contrast to the spatial limitations of multiphoton activation, single-photon activation results in an effective photon distribution across the entire z-axis of the illumination, so the power and duration of the laser illumination can be carefully controlled to minimize undesired activation of CHeX-seq oligonucleotides outside the desired activation region. Furthermore, ultrahigh-resolution activation in fixed cells can be achieved using evanescent waves from near-field fiber optics, providing 10-50 nm resolution.

[0630] To obtain a complete chromatin complement of a single cell, laser photoactivation must occur throughout the volume of the nucleus, which can be achieved by moving the photoactivation laser to different locations within the nucleus. If activation of CHeX-seq oligonucleotides occurs outside the nucleus, there should be essentially no effect, since the CHeX-seq polymerase is a DNA polymerase and will only perform DNA replication if DNA is the template. Therefore, CHeX-seq oligonucleotides annealed to RNA in the nucleus or cytoplasm will not be extended to produce complementary DNA.

[0631] In some embodiments, the reporter-tagged reversibly terminated oligonucleotide is reversibly terminated due to the presence of an enzyme-cleavable site, resulting in the removal of the non-extendable 3' end and the creation of an extendable 3' hydroxyl. The non-extendable 3' end can be a 3' end phosphate, a 2', 3'-cyclic phosphate, a 2'-ortho-methyl, a base modification, or a backbone sugar or phosphate modification. The cleavable site can be positioned toward the 3' end of the oligonucleotide. The cleavable site can be a specific sequence known as a target of an endonuclease. Incubation with an endonuclease can cause the annealed oligonucleotide to be cleaved at the site. The site can be a specific nucleotide, such as a cleavable base. As used herein, "cleavable base" refers to a nucleotide that is not usually present in a DNA sequence. For most DNA samples, deoxyuridine is an example of a cleavable base. Although the triphosphate form of deoxyuridine, dUTP, exists in living organisms as a metabolic intermediate, it is rarely incorporated into DNA. When deoxyuridine is incorporated into DNA, the resulting deoxyuridine is rapidly removed in vivo by normal processes, such as processes involving uracil-DNA glycosylase (UDG) (U.S. Pat. No. 4,873,192; Duncan, 1981; both references are incorporated herein by reference in their entirety). Thus, deoxyuridine rarely or never occurs in natural DNA. Non-limiting examples of other cleavable bases include deoxyinosine, bromodeoxyuridine, 7-methylguanine, 5,6-dihydro-5,6-dihydroxydeoxythymidine, 3-methyldeoxyadenosine, and the like (see Duncan, 1981).

[0632] The term "DNA glycosylase" refers to any enzyme with glycosylase activity, which causes the modified nitrogen-containing heterocyclic component of the nucleotide to be removed from the polynucleotide molecule, thereby generating an abasic site. DNA N-glycosylases include the following enzymes and their homologs in higher eukaryotes, including human homologs: uracil-DNA glycosylase (UDG), 3-methyladenine DNA glycosylase II (e.g., AlkA), TagI glycosylase, and MUG glycosylase. Uracil-DNA glycosylase recognizes uracil present in single-stranded or double-stranded DNA and cuts the N-glycosidic bond between the uracil base and the deoxyribose of the DNA sugar phosphate backbone, thereby leaving an abasic site. See, for example, U.S. Patent No. 6,713,294. The loss of uracil produces an abasic site in DNA. However, this enzyme cannot cut the phosphodiester backbone of the DNA molecule. Uracil-DNA glycosylases, abbreviated as "UDG" or "UNG", include mitochondrial UNG1, nuclear UNG2, SMUG1 (single-strand selective uracil-DNA glycosylase), TDG (TU mismatch DNA glycosylase), MBD4 (uracil-DNA glycosylase with methyl binding domain), and other eukaryotic and prokaryotic enzymes. Enzymes with this activity do not act on free dUTP, free deoxyuridine, or RNA. Another example of a UDG enzyme for generating one or more abasic sites is a thermostable homolog from Escherichia coli (E. coli) from Archaeoglobus fulgidus. Afu UDG catalyzes the release of free uracil from uracil-containing DNA. Afu UDG efficiently hydrolyzes uracil from single-stranded or double-stranded DNA. Another example includes Antarctic thermolabile UDG, which catalyzes the release of free uracil from single-stranded or double-stranded DNA containing uracil. The Antarctic thermolabile UDG enzyme is heat sensitive and is rapidly and completely inactivated at temperatures above 50°C.

[0633] Non-limiting examples of additional cleavable bases and their corresponding nicking agents are as follows: AlkA glycosylase recognizes and cleaves deoxyinosine residues; DNA-7-methylguanine glycosylase recognizes and cleaves 7-methylguanine residues; hypoxanthine-NDA glycosylase recognizes and cleaves hypoxanthine residues; 3-methyladenine-DNA glycosylase I (e.g., TagI) and 3-methyladenine-DNA glycosylase II (e.g., AlkA) recognize and cleave 3-methyladenine residues; Fpg recognizes and cleaves 8-oxo-guanine residues; and Mug recognizes and cleaves 3, N(4)-vinylcytosine and uracil residues from DNA.

[0634] As used herein, the term "abasic DNA" or "DNA with an abasic site" refers to a single-stranded or double-stranded DNA molecule that contains at least one abasic nucleotide, sometimes referred to as an "abasic site". "Abasic nucleotide" refers to a nucleotide that lacks a base in the 1' position of the deoxyribose sugar. As used herein, the term "AP endonuclease" or "AP lyase" refers to an enzyme that is capable of cleaving the phosphodiester backbone at an abasic site of a nucleic acid. The term includes enzymes that are capable of cleaving the 5' and 3' backbones of an abasic site.

[0635] The DNA sugar-phosphate backbone that remains after UDG cleavage of, for example, a glycosidic bond can then be cleaved, for example, by alkaline hydrolysis, high temperatures, tripeptides containing aromatic residues between basic residues (e.g., Lys-Trp-Lys and Lys-Tyr-Lys), and AP endonucleases (e.g., Endonuclease IV, Endonuclease V, Endonuclease III, Endonuclease VI, Endonuclease VII, Human Endonuclease II), etc. Thus, enzymes such as APE I can be used in conjunction with UDG to remove dU residues from nucleic acid molecules and then produce nicks in the nucleic acid molecules. Examples of enzymes for producing nicks at abasic sites include apurinic / apyrimidinic (AP) endonucleases such as APE 1 (also known as HAP 1 or Ref-1), which share homology with the E. coli exonuclease III protein. APE 1 cleaves the phosphodiester backbone immediately 5′ to the AP site via a hydrolytic mechanism to generate single-stranded DNA breaks, leaving behind a 3′-hydroxyl and a 5′-deoxyribose phosphate terminus.

[0636] Artificial nicking agents can be generated by combining a DNA N-glycosylase and an AP endonuclease, such as by combining a UDG glycosylase with an APE I endonuclease or combining an AlkA glycosylase with an EndoIV endonuclease, to achieve single-stranded cleavage at a cleavable nucleotide. Examples of nicking agents capable of excising modified nucleotides described herein include: (i) for excising deoxyuridine - a mixture of UDG glycosylase and EndoIV endonuclease; a mixture of UDG glycosylase and FPG glycosylase / AP lyase; a mixture of UDG glycosylase and EndoVIII glycosylase / AP lyase; a mixture containing UDG glycosylase, EndoIV endonuclease, and EndoVIII glycosylase / AP lyase; (ii) for excising 8-oxo-guanine and deoxyuridine - a mixture containing UDG glycosylase, FPG glycosylase / AP lyase, and EndoIV endonuclease; a mixture of UDG glycosylase and FPG glycosylase / AP lyase; and (iii) for excising deoxyinosine - a mixture of AlkA glycosylase and EndoVIII glycosylase / AP lyase, or a mixture of AlkA glycosylase and FPG glycosylase / AP lyase.

[0637] Endonuclease VIII from Escherichia coli acts as both an N-glycosylase and an AP lyase. The N-glycosylase activity releases degraded pyrimidines from double-stranded DNA, thereby producing abasic sites. The AP lyase activity cuts 3' into abasic sites, leaving 5' phosphate and 3' phosphate. The degraded bases recognized and removed by endonuclease VIII include urea, 5,6-dihydroxythymine, thyminediol, 5-hydroxy-5-methylhydantoin, uracildiol, 6-hydroxy-5,6-dihydrothymine, and methylpropanol diurea. Although endonuclease VIII is similar to endonuclease III, endonuclease VIII has β and δ lyase activities, while endonuclease III has β lyase activity.

[0638] Fpg (formamidopyrimidine [fapy]-DNA glycosylase) (also known as 8-oxoguanine DNA glycosylase) acts as both an N-glycosylase and an AP lyase. The N-glycosylase activity releases degraded purines from double-stranded DNA, thereby generating apurinic sites. The AP lyase activity cleaves both the 3′ and 5′ into apurinic sites, thereby removing the apurinic site and leaving a base vacancy. Some of the degraded bases recognized and removed by Fpg include 7,8-dihydro-8-oxoguanine (8-oxoguanine), 8-oxoadenine, fapy-guanine, methyl-fapy-guanine, fapy-adenine, aflatoxin Bl-fapy-guanine, 5-hydroxy-cytosine, and 5-hydroxy-uracil.

[0639] Also considered is called USER TM An enzyme nicking agent that specifically nicks the deoxyuridine portion of the target molecule; and USER TM Enzyme 2, which specifically nicks both deoxyuridine and 8-oxoguanine of the target molecule, leaving a 5' phosphate at both the nick site (see US Pat. No. 7,435,572). TM The enzyme is a mixture of uracil-DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII. UDG catalyzes the excision of the uracil base, thereby forming an abasic (apyrimidinic) site, while leaving the phosphodiester backbone intact. The lyase activity of endonuclease VIII cleaves the 3' and 5' sides of the abasic site of the phosphodiester backbone, thereby releasing the abasic deoxyribose.

[0640] After photoactivation, the 3′ end of the oligonucleotide can be extended in situ, allowing subsequent DNA polymerase-mediated complementary DNA synthesis using genomic DNA as a template ( Figure 2 ). To this end, DNA polymerase I can use the terminal 3' hydroxyl group of each annealed oligonucleotide to synthesize complementary DNA in situ from open genomic DNA. After DNA synthesis, the complementary DNA is dehybridized from the genomic DNA (for example, by heating the sample), removed, copied into double-stranded DNA, and amplified. Amplification can include PCR amplification, rolling circle amplification, or RNA amplification. In the case of PCR amplification, the CHeX-seq oligonucleotide can include a first primer binding site in its amplification segment, and a second primer binding site can be added to the 3' end of the extended cDNA by connecting a single-stranded primer sequence. In the case of RNA amplification, the CHeX-seq oligonucleotide can include, for example, a Sp6, T3, or T7 promoter sequence, and amplification is performed using Sp6, T3, or T7 RNA polymerase, respectively.

[0641] The amplified polynucleotides can then be made into cDNA libraries that can be sequenced, for example, using next generation sequencing. It is also possible to multiplex the CHeX-seq oligonucleotides by synthesizing multiple barcoded oligonucleotides that can be repeatedly diffused into fixed cells. This would allow the assay to be adapted for high throughput analysis.

[0642] III. Compositions and Methods for Detecting Cytoplasmic RNA (LT-TISA)

[0643] Molecular profiling of individual cells has described endogenous transcriptome variability within and between cells using a variety of techniques, including targeted amplification (Cornelison and Wold, 1997; Tay et al., 2010; Miyashiro et al., 1994), fluorescence in situ hybridization (FISH), and whole transcriptome assays. In addition to variability in expression levels, RNA sequencing from single cells has revealed heterogeneity in transcript forms, such as splicing products and 5′ sequences, between different cells (Dueck et al., 2015). This variability suggests that regulatory control processes governing the fate of individual cells may be obscure when measured using pools of cells, even if the cell phenotype is homogeneous. This individual cell variability may be required for the function of tissue-level systems (e.g., creating and maintaining neuronal diversity) (Dueck et al., 2016).

[0644] Over the past seven years, researchers have worked to create a versatile toolkit to perform genomic analysis on single living cells residing in natural microenvironments, which enables RNA analysis of living cells for studies where living tissue is available. However, for many human studies, living tissue is not available, so methods for analysis of transcriptomes in fixed cells must be developed and optimized. There are protocols for doing this starting with the use of in situ transcriptional methodology (Tecott et al., 1988; Crino et al., 1996; Miayshiro et al., 2003) and more recently Mer-FISH (Mofitt and Zang, 2016), Seq-FISH (Shah et al., 2017) and FISSEQ (Lee et al., 2015). In short, Mer-FISH allows iterative hybridization of oligonucleotide FISH probes that allow transcripts to be localized to many cells to be identified. Seq-FISH is similar to Mer-FISH, except that an iterative HCR (Choi et al., 2016; Choi et al., 2010) hybridization probe scheme is used, which provides a much stronger signal. Published Mer-FISH protocols allow the identification of hundreds of RNAs in cells, while Seq-FISH has been used to identify about 100 RNAs. As hybridization-based detection schemes, they cannot easily report splice variants or easily quantify the increase in background with each annealing. FISSEQ is a method for sequencing RNA directly in fixed tissue sections, where the RNA has been replicated in situ into cDNA (using in situ transcription technology (Crino et al., 1996)) and linearly amplified using rolling circle amplification. Due to the rolling circle amplification problem, the scheme is limited to about 400 RNAs. However, the first unbiased process in FISSEQ is in situ transcription, which requires oligonucleotides to hybridize with RNA to initiate cDNA synthesis, which can then be removed from fixed cells, amplified and sequenced. This article provides methods for modifying, optimizing and using in situ transcription to assess the RNA complement of fixed cells. This can be performed simultaneously with CHeX-seq in the same cell.

[0645] Although TISA can be performed with any oligonucleotide with a free 3′-OH to achieve specific cell or subcellular activation, it is preferred to activate oligonucleotides only at subcellular sites where cDNA synthesis is required. This can be achieved by using reversible termination nucleotides labeled with reporters, as discussed above in the context of CHeX-seq oligonucleotides. In this version of TISA, called LT-TISA, oligonucleotides can be synthesized that will bind to the poly A tail of RNA. In this way, the oligonucleotide can have an amplification segment (e.g., a primer binding site or RNA polymerase promoter) at the 5′ end, a specific barcode, and a poly dT segment (LT-TISA oligonucleotide) with a photoactivatable 3′ termination nucleotide at the 3′ end. In this case, the oligonucleotide is added to the fixed cell as an in situ hybridization oligonucleotide and anneals with the 3′ poly A tail of the single-stranded RNA in the cell (Tecott et al., 1988; Crino et al., 1996; Lee et al., 2015; Miyashiro and Eberwine, 2015). Alternatively, the LT-TISA oligonucleotides may contain a target-specific hybridization segment in place of the poly-dT segment. Thus, a set of LT-TISA oligonucleotides can be generated that specifically hybridizes with any desired batch of target RNAs. For cytoplasmic RNA characterization, the LT-TISA oligonucleotides are laser activated in the cytoplasm of fixed cells (thereby eliminating genomic DNA contamination), and reverse transcriptase is added so that the activated LT-TISA oligonucleotides initiate cDNA using the annealed RNA as a template.

[0646] In some embodiments, this iteration of TISA will be multiplexed so that multiple cells can be analyzed individually by synthesizing LT-TISA degenerate sequence oligonucleotides with different barcodes (BC) (also used to multiplex cells, see below). For example, LT-TISA oligonucleotides with BC1 are annealed to fixed cells and activated in the cytoplasm of a single cell (or cell type, such as inhibitory neurons), and unactivated LT-TISA oligonucleotides are removed from other cells by optical washing (see discussion of optical washing). Next, a second LT-TISA oligonucleotide with BC2 is annealed to the cell and activated only in the cytoplasm of the second cell. Once the desired number of cells has been iterated, reverse transcriptase is added and cDNA is synthesized by extending the 3′ hydroxyl and using the annealed RNA as a template. After cDNA synthesis, all cDNAs can be removed by alkaline denaturation, amplified simultaneously, and made into sequencing libraries. The transcriptome of each cell is uniquely identified by the presence of BC on the reads associated with a specific subcellular region.

[0647] The LT-TISA method can be optimized to simultaneously detect pools of RNA from different subcellular regions by testing cell fixation conditions (type of fixative, fixation time, etc.), photoactivation parameters (optimal wavelength determination, energy requirements), and molecular biology procedures, thereby creating subregion-specific TISA libraries.

[0648] IV. Methods for Detecting Open Genomic DNA and Cytoplasmic RNA in Single Cells

[0649] The present invention provides methods for simultaneously quantifying the landscape of open genomic DNA in fixed cells and their cytoplasmic localized transcriptome. Previous methods for studying the landscape of open genomic DNA in cells require organelle separation, such as isolating the nucleus to perform Nuc-seq to assess nuclear RNA surrogates of cytoplasmic RNA or performing ATACseq for open chromatin analysis, but at the expense of cellular spatial location. Existing hybridization techniques, such as Mer-FISH or SeqFISH, retain spatial information but only allow for the assessment of a subset of cytoplasmic RNA. In addition, these methods do not allow for the characterization of chromatin and RNA from the same cell while maintaining tissue-related spatial resolution.

[0650] In order to understand how cells respond to their local environment, it is necessary to assess not only the abundance of cytoplasmic RNA, but also the structural dynamics of chromatin, which produces RNA. In order to ultimately provide information, these measurements must occur in the same cell so that the dynamic processes that affect the flow of information from chromatin to cytoplasmic RNA can be assessed without the dilution effect of other non-responsive cells or differently responsive cells. Quantitative assessment of all three in the same cell can be used to provide a detailed view of the transcriptional regulatory pathway, which can be used to manipulate the pathway to enhance the cell's response to various local stimuli, including those that cause disease.

[0651] Furthermore, while openness is necessary for transcription to occur, it is important to see the product of transcription, the cytoplasmic RNA, to assess how openness correlates with cytoplasmic RNA abundance. Simultaneous measurement of chromatin dynamics and cytoplasmic RNA populations in the same cell will detail the fine-tuning of transcriptome regulatory pathways across co-regulated genes.

[0652] Thus, methods are provided to coordinate the LT-TISA procedure with the CHeX-seq procedure, both of which use in situ cDNA synthesis and aRNA amplification, so that chromatin landscape determination and cytoplasmic RNA pool characterization can be accomplished in single fixed cells.

[0653] Because, in some embodiments, CHeX-seq will be performed first with a DNA polymerase, it may be preferable to remove the unactivated CHeX-seq oligonucleotides before performing the LT-TISA, so that the LT-TISA oligonucleotides can anneal to the RNA. Any unactivated CHeX-seq oligonucleotides will be removed using, for example, heat denaturation, so that the annealed 16-base short sequence will be dehybridized, while the DNA primed by the longer double-stranded CHeX-seq oligonucleotide will remain annealed. This will allow the extended CHeX-seq oligonucleotide to remain associated with the chromatin until after the subsequent LT-TISA reaction.

[0654] In some embodiments, optical wash protocols and on-microscope multiplexing of CHeX-seq and LT-TISA analysis can be performed on immune-specified cells. The estimated time for performing CHeX-seq and LT-TISA oligonucleotide addition and optical washes to place specific barcoded oligonucleotides in the nucleus and cytoplasm of multiple individual cells is 1 minute per barcoded oligonucleotide population. This suggests that 300 individual immune-identified cells with different barcodes in the nucleus (CHeX-seq) and cytoplasm (LT-TISA) can be completed in 10 hours. This can be accelerated, for example, by using a multi-barrel pipet spritzer with six barrels, so that six different oligonucleotides can be applied in a spatially restricted manner, thereby targeting 1,800 cells in 10 hours, each with a different barcoded oligonucleotide in the nucleus and cytoplasm.

[0655] V. Methods for analyzing the 3D structure of open genomic DNA around selected genes

[0656] This article provides a method for the structural analysis of chromatin niches around specific genes. This method allows the identification of promoter regulatory mechanisms in vivo, including enhancers and other genomic / chromatin regulatory regions that regulate selected genes locally in spatially defined single cells, because such regulatory elements are expected to be close to the regulated genes. Since chromosome packaging can locate genes from distant chromosomal regions (including different chromosomes near the target gene), it is important to define the chromosomal region near the target gene. The chromatin landscape of the genome used for expression and the 3D "chromatin niche" around a specific gene may vary from cell to cell, but are more similar in cells living in similar microenvironments. In addition, monitoring of chromatin niche dynamics over time can be used to detail how these important regulatory niches change over time and respond to external stimuli.

[0657] In this gene-specific iteration of CHeX-seq, fixed cells / tissues can be used for fluorescent in situ hybridization to detect the location of specific genes. This specific gene FISH signal will be used to target the nucleus using multiphoton activation of CHeX-seq oligonucleotides only at the gene where FISH is focused. For example, FISH probes directed to open genomic DNA regions near the transcription start site (TSS) of a selected gene can be used to generate a fluorescent signal at the TSS of the gene, which can be imaged. These probes can be directionally meaningful so that they anneal to genomic DNA sequences but not to RNA. CHeX-seq oligonucleotides can then be added, which will diffuse throughout the fixed cells / tissue and bind to open regions of cellular chromatin. In order to specifically assess the chromatin structure around the TSS of a selected gene, the FISH probe signal will be used to guide multiphoton irradiation to locally activate the CHeX-seq oligonucleotides at the TSS site of the gene ( Figure 6 ). CHeX-seq oligonucleotides activated near a selected gene can be extended to perform cDNA synthesis at a single-stranded open genomic DNA site near the TSS of the gene. This gene niche-specific CHeX-seq procedure can identify sequences corresponding to chromatin regions near the FISH probe site, which can be genomic regulatory elements (including enhancers) of transcription of the selected gene. These methods can be used to study any specified genomic DNA site. In addition, the use of optical cleaning can allow these methods to have higher yields.

[0658] VI. Multiplexing

[0659] As provided herein, methods for characterizing chromatin and cytoplasmic RNA can transition to medium / high throughput data generation platforms. In order to make the analysis of chromatin and cytoplasmic RNA a medium / high throughput procedure, methods are provided that allow iterative addressing of specific oligonucleotides. In Mer-FISH and Seq-FISH, this is achieved by chemical dehybridization and more recently photobleaching. However, these methods are not sufficient to discriminately remove unactivated oligonucleotides from between and within cells for subcellular analysis.

[0660] Therefore, methods for optical cleaning are provided herein. These methods use infrared (IR) lasers to locally heat solutions on fixed tissue sections to denature undesirable oligonucleotide binding. This is useful because the oligonucleotides used for CHeX-seq and LT-TISA are short and their hybridization can be easily controlled. In order to hybridize in the target cells, oligonucleotides with selected barcodes can be locally applied to the limited space on the fixed cells or in the tissue using a fully automatic microsprayer and aspiration system. When CHeX-seq and LT-TISA oligonucleotides hybridize and become fluorescently labeled by adding fluorescently labeled photoactivatable terminators (e.g., luminescent terminators), the target cells can be identified by immunofluorescence, and the oligonucleotide hybridization in the cell can be monitored by an increased fluorescence signal. Depending on the size of the oligonucleotide, temperature manipulation can be optimized, and the theoretical irradiation and heating area can be estimated by Fourier's law of heat conduction (p = -Ak (dT / dL), p = thermal power, A = heating area, k = thermal conductivity, dT = temperature gradient, and dL = distance). For example, for CHeX-seq oligonucleotides with a degenerate annealing sequence of 15 bases, the Tm range will be 35-42°C, and for LT-TISA (15 base poly A) oligonucleotides, the Tm is 35°C. Heating the solution surrounding the annealed oligonucleotides to 50°C will dehybridize >95% of the inactivated oligonucleotides. A temperature of 50°C can be reached instantaneously at the focused pixel using 30 mW of energy. The efficiency of dehybridization and oligonucleotide removal (e.g., by washing) can be easily monitored by a decrease in fluorescence signal. However, these methods are also applicable to situations where a microinjector is not available—bath application of oligonucleotides to the entire tissue can be performed (however, oligonucleotide hybridization will be slower), and denaturation of all areas except the cells of interest can be achieved by optical washing based on immunofluorescence image masking, which protects areas from heating, allowing selective heating of unstained cells ( Figure 7 ). This process can be incorporated into custom imaging software.

[0661] Within these experimental paradigms, there is considerable flexibility in the future to target different cellular compartments to achieve multiplexity, for example to assess the initial rapid nuclear transcriptional response as well as steady-state cytoplasmic RNA abundance in selected cells that are synaptically connected to each other in their natural microenvironment (tissue sections) (de la Torre-Ubieta and Bonni, 2011; Spaethling et al., 2017), and to analyze different RNA populations (e.g., mRNA, noncoding RNA, total RNA).

[0662] VII. Exemplary Applications of the Methods of the Invention—Chromatin Remodeling in Neurodevelopmental and Psychiatric Disorders

[0663] The pharmacology of chromatin remodeling is relatively understudied when compared to its important role in neurodevelopment and age-related plasticity (Borrelli et al., 2008; Ziller et al., 2015). In neurons, an important regulator of plasticity in these biological processes is BDNF, whose effects on neuronal differentiation and local protein synthesis have been well documented (Martinowich et al., 2003; Berton et al., 2006). Other plasticity phenomena that have been associated with chromatin changes include the effects of strong depolarizing stimulation on dendritic morphology (Ellis et al., 2016; de la Torre-Ubieta and Bonni, 2011; Seifuddin et al., 2013). In genome-wide association studies (GWAS) in autism spectrum disorder (ASD) and in chronic alcohol addiction, chromatin remodeling and changes in dendritic morphology have been associated with activation of NMDA glutamate receptors, voltage-sensitive Ca++ channels, and GABAb receptors (Varodayan and Harrison, 2013; Costa et al., 2006; Guidotti et al., 2011), which mediate hyperpolarization under normal conditions. These data on chromatin remodeling come from the analysis of a large number of cells.

[0664] Genome-wide association studies have identified multiple loci associated with schizophrenia and depression, but many of these loci have not yet provided mechanistic insights into disease causality (Maurano et al., 2012). Therefore, it is important to think beyond the idea of ​​genetics and focus on how the genome is regulated (Insel and Wang, 2010). There is growing evidence that cells differ significantly in their susceptibility to diseases such as depression, and therefore respond differently to treatment. Transcriptome variability has been observed in schizophrenia (Zhang et al., 2015) and depression (Roussos et al., 2014), including noncoding RNA populations. The RNA transcription process requires DNA in a specific conformation (i.e., open), in which there is less nucleosome packaging, so that transcriptional regulatory enzymes can function. Chromatin variations between neurons and other cell types have also been noted (Fullard et al., 2017), specifically including differences in genomic DNA methylation between neurons and astrocytes (Kozlenkov et al., 2014; Kozlenkov et al., 2016). Such epigenetic methylation differences have also been observed between cells of other cell types (Mo et al., 2015). There have been some efforts to link transcriptome and chromatin variability, arguing that having both sets of data will enable a better understanding of the regulatory mechanisms that underlie cellular dysfunction in these diseases (Xiao et al., 2014). The plasticity of chromatin responsiveness has been highlighted in developmental changes (Zhu et al., 2013), pharmacological manipulation of cocaine (Kumar et al., 2005), and changes in mood states (Renthal et al., 2007). These studies have shown chromatin variation between cell types and subtypes, even from enriched populations of cells. Such variation highlights the need to assess this variation at the single-cell level, where mechanistic insights are more easily obtained. The literature reporting on chromatin reorganization almost exclusively uses acute and robust pharmacological manipulations of cultured cell lines—there is limited exploration of more subtle regulatory effects of synaptic transmitters (which have clinical implications) or their pharmacological equivalents (such as receptor or channel modulators) in slice preparations. To address the question of how psychoactive drugs affect chromatin structure around specific genes, one needs to be able to assess chromatin structure locally at these subgenomic single-gene loci (Kolovos et al., 2014; Heller et al., 2014).

[0665] The molecular effects of synaptic stimulation on chromatin remodeling follow established receptor- and calcium-binding protein-mediated pathways (Frankle et al., 2003). In neurons from the mouse hippocampus and cerebral cortex, chromatin remodeling has been mechanistically linked to the chromatin remodeling proteins AXTR, HSF1, and H3.3 (Varodayan and Harrison, 2013). These proteins are also affected by 5HT receptor-mediated stimulation, which is implicated in mood disorders. This links mood disorders to other psychiatric disorders via a chromatin remodeling mechanism (Duman, 2013; Sun et al., 2015; McCarthy et al., 2014). Mood disorders are therapeutically affected by the most popular antidepressants, the SSRIs, and similarly, synapse-mediated fine-tuning has been observed with the most commonly used antipsychotics, the D2 antagonists (Vialou et al., 2013).

[0666] Background on transcriptome variability: Recent technological advances have enabled increasingly high-resolution measurements of gene expression in single cells, leading to a growing appreciation of the extent of expression variability between cells. This variability has been examined as: 1) an indicator of the vast diversity of cell types in multicellular organisms; 2) a byproduct of redundancy in regulatory networks; 3) a temporal snapshot of asynchronous dynamic processes; 4) a product of molecular dynamics; or 5) as evidence that RNA abundance may not correlate with cellular phenotype. An alternative perspective is to consider whether single-cell transcriptome, proteome, and other molecular variability is critical for function at the level of the cell population. Are individual cells in a multicellular tissue analogous to individual organisms in a cooperative community, where the behavior of each cell contributes to the production of a higher-level overall function?

[0667] This variability reflects the many-to-one relationship between transcriptome state and cell phenotype (Kim and Eberwine, 2010). In this relationship, the molecular ratio of RNA subsets is determined by the stoichiometric constraints of the cell system, and the stoichiometric constraints insufficiently illustrate the transcriptome state. By analogy, the transcriptome phenotype is defined as the RNA subset that constitutes the selected RNA system, which exists in a balance with each other to produce associated cell functions. There is considerable evidence that individual cells in tissues take heterogeneous states by relaxing their physiological dynamics or by active signaling and maintaining an aggregated state.

[0668] Furthermore, if gene expression variation between individual cells is important for tissue function, the degree of variation itself may be conserved across species. As a proof of principle, F statistics were calculated for cortical and hippocampal pyramidal neurons in rats and mice. For each cell type, partial correlations of the F statistics across species were calculated, controlling for gene expression levels to ensure that correlations were not simply due to shared levels of gene expression. The partial correlation coefficients across species were significant for both cell types examined (two-sided T-test of association p<10 -13 , Figure 8 ), indicating conservation of gene expression variation across species. These data support the hypothesis that, at least for some genes, gene expression variation is regulated and that expression patterns across cell populations may be important for tissue function.

[0669] Provided herein are methods that enable determination of whether the positioning of chromosomes relative to cellular signaling inputs (due in part to the polarity and orientation of cells) generates cellular transcriptome variability and promotes a "many-to-one" elaboration of similar cellular physiological states (Kim and Eberwine, 2010).

[0670] Effects of neuronal morphological geometry on the nucleus: CNS neurons are located in brain structures where they receive synaptic input from many presynaptic neurons. During neuronal development, neurons migrate to a location where they become "hardwired" or synaptically and spatially interconnected with surrounding cells ( Fig. 9 , top). These neurons can change their synaptic connectivity, but they have limited ability to move or reposition themselves. The nucleus in the neuron is relatively immobile, with the neuronal cytoskeleton association holding the nucleus in place. Because these neurons are postmitotic, there is little noticeable movement of the chromosomes, e.g., chromosome 1 cannot drastically change position with chromosome 5 ( Fig. 9 , middle). However, cis- and trans-regulatory regions that control chromatin accessibility and expression do move. Any two adjacent cells may have overlapping higher-order chromatin structures, resulting in common cellular features as well as variable structures that lead to cellular heterogeneity.

[0671] Used to create a model of transcriptome variability: This morphological constraint suggests that activation occurs in a vectorial manner from the site of stimulation as different neurons receive input from the presynaptic neuron. When the signaling cascade reaches the nucleus, the open chromatin regions closest to the site of stimulation will receive the strongest and fastest stimulation ( Fig. 9, where neuron A has green and purple chromatin on the side of the nucleus closest to the stimulus; neuron B has black and purple chromatin; and neuron C has blue and green chromatin). Given the predicted differences in chromosome localization, this further suggests that chromatin interactions also differ to a large extent due to different enhancer and target gene interactions in selected cells. Subsequently, differences in the proximity of vector stimuli and differences in chromosome / chromatin interactions will lead to transcriptional variability ( Fig. 9 , bottom small image).

[0672] The methods provided herein can be used to identify changes in chromatin state and / or cytoplasmic transcriptome caused by clinically relevant manipulations (e.g., antidepressant effects) (Tsankova et al., 2007; Sharma et al., 2006; Ellis et al., 2016). Clinical depression is a psychiatric disorder that can be difficult to treat because long onboard times are required for drugs to be effective and dosage adjustments are required to be effective. The molecular basis of these problems may be partly due to the genomic variability that exists between cells and the need to change the epigenetic state of the adult neuronal genome. To this end, drugs for the treatment of depression can be tested for their ability to regulate chromatin structure and cytoplasmic RNA abundance in the context of variability between cells. Because "cell-to-cell" differences in chromosome organization may be the basis of cell-to-cell variability in the transcriptional response to antidepressants, analysis of the 3D structure of open chromatin around selected genes can provide further insight. This knowledge may promote the development of new methods for manipulating higher-order chromatin structures to enhance the therapeutic efficacy of these drugs.

[0673] To this end, the CHeX-seq protocol can be used to generate single-neuron open chromatin datasets of dispersed mouse and human cortical neurons. In addition, the combined CHeX-seq and LT-TISA protocols can be used to generate quantitative single-neuron transcriptional biology datasets based on open chromatin analysis of steady-state levels of cytoplasmic RNA pools in dispersed mouse and human cortical neurons. Simultaneous measurement of multiple cytoplasmic RNA pools with chromatin structure can show which regions of transcriptional pathways are most closely associated with drug responsiveness to antidepressants. Finally, the time course of chromatin niche remodeling around specific genes will provide the first insight into how conserved chromatin remodeling processes are for genes associated with psychiatric disorders. This information will be useful for assessing the functional interaction between the timing of niche development and phenotypic outcomes.

[0674] Specifically, the methods of the present invention can be used to assess the "regulated transcriptome" effects of chromatin remodeling in single-cell cortical neurons in response to the antidepressant fluoxetine. Simultaneous measurement of multiple cytoplasmic RNA pools with chromatin structure can be used to reveal which regions of transcriptional pathways are most closely associated with drug responsiveness to antidepressants (Tsankova et al., 2007). As a corollary to these genome-level responses, the time course of chromatin niche remodeling around specific genes can be examined to provide the first insight into how chromatin remodeling processes are conserved for genes associated with psychiatric disorders. This information will be useful for assessing the functional interactions between the timing of niche development and phenotypic outcomes. Single-gene chromatin niche analysis can focus on the pharmacological modulation of chromatin structure around genes encoding chromatin remodeling proteins such as AXTR, HSF1, and H3.3 (which must be active during remodeling), as well as pharmacologically responsive target genes, including members of the glutamate and GABAb receptor and CREB second messenger activation systems, which are involved in GPCR-mediated cellular events (Ruzicka et al., 2015; Huang and Akbarian, 2007; Chen et al., 2014).

[0675] An experimental system often used to evaluate the biological mechanism of certain cells is the primary dispersed CNS cell culture. Long-term (2-3 months) mouse culture requires neurons from fetuses / newborn mice, because mature neurons cannot usually remain viable for a long time in culture. Short-term mouse primary cell culture from adult brain can be completed rapidly in up to 3 days. In the past, it was believed that human neuronal cells were also the same, but in order to test this, the inventors have collected living human brain tissue from patients and characterized it. Brain tissue is obtained from consenting patients who undergo neurosurgery (usually for tumor removal). Laboratory staff wait during the surgical procedure and usually send suitable tissue to the laboratory within 20 minutes of resection. Cells have been enzymatically dissociated from a small amount of tissue, and dispersed adult neuron primary cell culture has been successfully performed for 3 months and longer. Primary cell culture models can be used to predict and separate factors involved in various physiological events, including the interaction of RBP and mRNA, which can be tested in vivo subsequently. The utility of these long-term human cell cultures is reflected in the tendency of cells of specific types to reveal their different categories only within a subset of genes. For example, RNAseq analysis of approximately 300 human brain-derived cells followed by pathway analysis showed the presence of neurons, astrocytes, endothelial cells, microglia, and oligodendrocytes in human brain cell cultures. Identifying subcell types from single-cell samples requires careful application of noise control, pathway / gene selection, and machine learning methods. These data indicate that the human primary cell culture method is applicable to a variety of transcriptionally defined cell types, including neurons that will be used in these studies (Thurman et al., 2012).

[0676] VIII. Kit

[0677] The kit according to the present invention provides at least one component that can be used for practicing at least one embodiment of the inventive method. Therefore, the kit can provide some or all components necessary for practicing at least one embodiment of the method according to the present invention. The kit may include at least one container in which the oligonucleotide of the present invention is housed. The kit may include all oligonucleotides required for performing at least one embodiment of the method according to the present invention, such as a group of open genomic DNA and / or expressed oligonucleotides for analyzing a selected gene, or a group of degenerate oligonucleotides for the full genome analysis of open genomic DNA.

[0678] Test kit is generally defined as a package comprising one or more containers, which contain one or more oligonucleotides or compositions of the present invention. The test kit itself can be made of any suitable material, including but not limited to cardboard, metal, glass, plastic or some other polymeric materials known to be used for packaging and storing biological samples, research reagents or materials. The test kit can be designed to keep one or more containers, each of which is designed to keep one or more nucleic acids, compositions or samples of the present invention. The container can be made of any suitable material, including but not limited to glass, metal, plastic or some other suitable polymeric materials. Each container can be selected independently for material, shape and size. Non-limiting examples of containers include pipes (e.g., microcentrifuge tubes), vials, ampoules, bottles, jars, bags, etc. Each container can be sealed with a permanent seal or a reclosable seal (e.g., screw cap). One or more containers in the container in the test kit can be sterilized before or after being included in the test kit.

[0679] The kit of the present invention may include one or more other components or materials that can be used for practicing the inventive method, such as sterile water or aqueous solution, a buffer for performing the various reactions involved in the inventive method, and / or a reagent for detecting an amplified product. Therefore, the kit may include one or more polymerases for amplifying cDNA or RNA molecules. The kit may include one or more reverse transcriptases for synthesizing cDNA from an RNA template. According to an embodiment of the present invention, it may also include some or all of the components, reagents and supplies for performing reverse transcription and amplification. For example, in some embodiments, in addition to comprising one or more oligonucleotides of a photoactivatable terminator, the kit may also include nucleotides for reverse transcription and / or amplification, with or without one or more polymerases. In an embodiment, it includes some or all of the reagents required for library preparation and next generation sequencing analysis.

[0680] IX. Examples

[0681] The following examples are included to illustrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention and therefore can be considered to constitute preferred modes of the practice of the present invention. However, it will be appreciated by those skilled in the art based on this disclosure that many changes may be made to the disclosed specific embodiments and still obtain the same or similar results without departing from the spirit and scope of the present invention.

[0682] Example 1 - CHeX-seq oligonucleotide terminator addition

[0683] HPLC purified oligonucleotides and their complementary oligonucleotides were purchased from Integrated DNA Technologies (IDT). A template-dependent DNA polymerase binding assay was used to bind fluorescently labeled photoactivatable terminator nucleotide analogs to the 3′ end of the oligonucleotides: (1) 5 μM oligonucleotide, 25 μM complementary oligonucleotide, 50 μM fluorescently labeled photoactivatable terminator nucleotide analogs, 4 mM MgSO4, and 0.1 U / μL Therminator (New England Biolabs) were mixed in 1× ThermoPol buffer, (2) the mixture was heated to 80°C for 45 seconds, and (3) the mixture was incubated at 60°C, 55°C, 50°C, 45°C, 40°C, 35°C, 30°C, and 25°C for 5 minutes each. The binding product was purified on an Agilent 1260 Infinity reverse phase HPLC using an XTerra MS C18 preparative column (Waters). The purified product solution volume was concentrated to about 250 μL using an Eppendorf Vacufuge and then denatured into single-stranded oligonucleotides using an equal volume of 0.2 M NaOH. HPLC purification and concentration were repeated using the same conditions for collecting oligonucleotides. The final product was dissolved in 1x PBS and the concentration was determined by measuring the fluorescent dye absorbance.

[0684] Example 2 - CheX-seq

[0685] CHeX-seq was performed using cells fixed in 3% formalin for 3 minutes.For this experiment, CHeX-seq oligonucleotides targeting single nuclei of cortical neurons were used. Fig.10 A summary of alignments of reads from individual cells is shown. These alignments are plotted as being associated with different genomic features, transcription start sites (TSS), coding sequences (CDS), 3′UTRs, and intergenic regions. The read results show peaks at the expected positions close to the TSS and across the coding sequence. Interestingly, peaks can also be seen at both ends of the intergenic regions.

[0686] Materials and methods for Examples 3-4-7

[0687] Human brain tissue. Human brain tissue was collected at the Hospital of the University of Pennsylvania (IRB No. 816223) using standard operating procedures for recruitment and informed consent of patients. Briefly, whole brain samples (usually 5×5×5 mm) were obtained from the cortex, which was removed as part of neurosurgery for the treatment of epilepsy or brain tumors. The tissue was immediately transferred to a container containing ice-cold oxygenated CSF (in mM: KCl is 3mM, NaH2PO4 is 2.5mM, NaHCO3 is 26mM, glucose is 10mM, MgCl2-6H2O is 1mM, CaCl2-2H2O is 2mM, sucrose is 202mM, and a gas mixture containing 5% CO2 and 95% O2) for transfer to the laboratory. The tissue arrived at the laboratory approximately 10 minutes after resection. The brain tissue was then processed for cell culture and fixation.

[0688] Cell culture / preparation and fixation. K562 cells were obtained from ATCC and cultured in RPMI 1640 medium (Invitrogen) containing 10% FBS and penicillin-streptomycin in T75 flasks at 37°C for 2-3 days in 5% CO2. The cultured cells were transferred to a 50 ml tube and fixed by adding 16% paraformaldehyde (final 1%) for 10 minutes at room temperature. After fixation, quenching was performed using 1M glycine (final 200mM) containing RPMI 1640 medium for 10 minutes, followed by centrifugation at 300xg for 5 minutes. The supernatant was discarded and 3 mL of PBS was added to the pellet, which was then mixed by gently pipetting up and down 10-15 times using a fire-polished glass pipette to prevent cell clumping and centrifuged at 300 rpm for 5 minutes. 100 μl of the cell pellet was attached to an 18 mm gridded coverslip by incubation for 2 hours at room temperature. The samples were washed with PBS containing 0.01% Triton X-100 (without Ca ++ Mg ++ ) for 10 min, and then washed with PBS (without Ca ++ Mg ++) were washed three times for 3 minutes each. To prepare human neuronal cell cultures, adult brain tissue was placed in a papain (20U, Worthington Biochemical) solution for dissociation at 37°C for 30 to 40 minutes, and then the enzymatic dissociation was stopped with ovomucoid (papain inhibitor, 10 mg / ml, Worthington Biochemical) (Spaethling et al., 2017). The tissue was ground with a fire-polished Pasteur glass pipette. The turbid cell suspension was carefully transferred to a new test tube and centrifuged at 300×g for 5 minutes at room temperature. The cells were counted in an automatic counter (Invitrogen). The cells were counted at 3×10 4 The density of 10 cells / coverslip was seeded on poly-L-lysine coated (0.1 mg / ml, Sigma-Aldrich) 12 mm coverslips. The cultures were incubated at 37°C, 95% humidity and 5% CO2 in neuronal basal medium (Neurobasal A, Gibco), serum-free supplement (B-27, Gibco) and 1% penicillin / streptomycin (Thermo-Fisher Scientific). Dispersed mouse neuron / astrocyte cultures were prepared according to published protocols (Buchhalter and Dichter, 1991). The dispersed cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. After this, they were washed three times with 1×PBS. The cells were permeabilized with 0.1% Triton-X100 for 10 minutes at room temperature and then washed three times with 1×PBS.

[0689] Mouse brain tissue slice preparation. 3-month-old male mice were anesthetized with halothane, euthanized by thoracotomy, and then perfused with 5 ml PBS followed by 20 ml PBS / 4% paraformaldehyde. The brain was removed and fixed for 16 hours at 4°C, then rinsed with PBS and sectioned at 100 μm in the coronal plane on a vibratome (Leica VT-1000s). The slices containing the hippocampus were then immunofluorescently labeled with chicken anti-MAP2 antiserum (1:1000; Ab 5392; Abcam) followed by Alexa 488-conjugated goat anti-chicken secondary antibody (1:400; ab150169; Abcam).

[0690] CHeX-seq probe synthesis: HPLC-purified probe oligonucleotides and their complementary oligonucleotides were purchased from Integrated DNA Technologies (IDT). The Cy5 dye-labeled luminescent terminator was synthesized using a template-dependent DNA polymerase binding assay. TM (Lightning Terminator TM) (Agilent, Inc.) to the 3′ end of the probe oligonucleotide: (1) 5 μM probe oligomer, 25 μM complementary oligomer, 50 μM Cy5-labeled luminescent terminator TM , 4 mM MgSO4, and 0.1 U / μL of Therminator (New England Biolabs) in 1× ThermoPol buffer, (2) the mixture was heated to 80°C for 45 seconds, and (3) then incubated at 60°C, 55°C, 50°C, 45°C, 40°C, 35°C, 30°C, and 25°C for 5 minutes each. The conjugated product was purified on a 1260 Infinity reverse phase HPLC (Agilent Technologies) using an XTerra MSC18 preparative column (Waters). The purified product solution was concentrated to approximately 250 μL using a Vacufuge (Eppendorf) and then denatured into single-stranded oligomers using an equal volume of 0.2 M NaOH. HPLC purification and concentration were repeated using the same conditions for collecting the luminescent terminator-labeled single-stranded probe. The final product was dissolved in 1× PBS and the concentration was determined by measuring the Cy5 absorbance at 647 nm (Figure S1).

[0691] Application of CHeX-seq probes. After fixation and permeabilization, cells and brain sections were incubated with CHeX-seq probes (170 nM) in TES buffer (10 mM Tris, 1 mM EDTA, 150 mM NaCl) for 1 h at room temperature. ++ Mg ++ ) Wash the samples 3 times, 3 minutes each time.

[0692] Imaging and Photoactivation. After CHeX-seq probe annealing and washing, samples were transferred to PBS containing 1× PBS (without Ca ++ Mg ++ ) in an imaging chamber. All imaging and photoactivation were performed using a Carl Zeiss 710Meta confocal microscope (20x water immersion objective, NA 1.0). CHeX-seq probe annealing was confirmed by excitation at 633nm, and emission was detected at 640-747nm. Photoactivation was performed using a 405nm (UV) laser at 60% power and 6.30μs per pixel.

[0693] First-strand DNA in situ synthesis and single-cell harvesting. After photoactivation in each individual cell nucleus, a master mix containing DNA polymerase I and first-strand DNA synthesis buffer was added to the cells and incubated for 1 hour at room temperature. Subsequently, single cells containing synthesized complementary DNA were harvested using a glass micropipette for visualization under a Zeiss 710 confocal microscope (Carl Zeiss).

[0694] Linear amplification of nucleosome-free regions of chromatin. (A) First-strand DNA synthesis and poly-G tailing at the 3′ end: After harvesting single cells, cDNA synthesized in situ was removed by adding freshly prepared 0.1N NaOH and incubating the samples at room temperature for 5 minutes, followed by neutralization with 1M Tris (pH 7.5). After ethanol precipitation, the first-strand DNA was resuspended in nuclease-free water. Subsequently, poly(G) was added to the 3′ end using terminal deoxynucleotidyl transferase (TdT) (Invitrogen). (B) Second-strand DNA synthesis and first-round linear RNA amplification: After priming with a custom App-RC-polyC primer (Table 1), second-strand DNA was synthesized using DNA polymerase I at 16°C for 2 hours. Linear in vitro transcription was performed from the T7 RNA polymerase promoter incorporated into the double-stranded DNA using the Ambion MEGA Script T7 In Vitro Transcription (IVT) kit to amplify RNA. (C) Second round of first- and second-strand DNA synthesis and PCR amplification: After cleanup of the IVT reaction, first-strand DNA was reverse transcribed from aRNA using superscript III using custom App-RC primers (Table 1). Second-strand DNA was synthesized using DNA polymerase 1 and custom 18bpPBC1 primers (Table 1). Subsequently, double-stranded blunt-end DNA was amplified using custom primers 18bpPBC1 / App-RC (Table 1) under the following polymerase chain reaction conditions: 98°C for 30 seconds; thermal cycling at 98°C for 10 seconds, 50°C for 30 seconds, and 72°C for 30 seconds for 27 cycles; extension at 72°C for 2 minutes, and then used for library construction. Samples for control experiments were processed with the same procedure, except that no CHeX-seq probe was applied, and a second round of second-strand DNA PCR amplification was performed using custom primers 18bpPBC14 / App-RC (Table 1).

[0695] Table 1. List of sequences of primers and oligonucleotides used in these studies.

[0696]

[0697] Sequencing library preparation. The Illumina TruSeq Nano DNA Library Preparation Kit was used with modifications. All the double-stranded DNA amplified in the second round of PCR was used as input. After the DNA fragments were converted to blunt ends with an end repair mixture, the base "A" was added; the sequence adapter was connected. The DNA insert was amplified by PCR.

[0698] External data. GRO-seq: K562 GRO-seq was downloaded in FASTQ format from SRA (accession number GSE60454) (Core et al., 2014); raw reads were processed using the SCAP-T pipeline (available on the World Wide Web at scap-t.org); POL2-engaged transcripts were inferred by HOMER (Heinz et al., 2010); ATAC-seq: 1. Single-cell unprocessed K562 ATAC-seq data was downloaded in raw FASTQ format from SRA (accession number GSE65360) (Buenrostro et al., 2015). The alignment and peak calling methods in Buenrostro et al. (2015) were followed; 2. Mouse brain ATAC-seq data were downloaded from ENCODE (Davis et al., 2018) in BAM format; narrow and broad peaks were called using MACS2 (Feng et al., 2012); DNAse-seq: 1. K562 DNAse-seq narrow and broad peaks were downloaded from ENCODE in bigBed format; 2. Human brain DNAse-seq data were downloaded from ENCODE in BAM format; FAIRE-seq: K562 FAIRE-seq narrow peaks were downloaded from ENCODE (accession number ENCFF000TLT) in BED format; the original hg19 genome build was promoted to hg38 by CrossMap (Zhao et al., 2014); Reduced representation bisulfite sequencing (RRBS): K562 was downloaded from the UCSC ENCODE track in BEDMethyl format DNA methylation RRBS data; the original hg19 genome build was promoted to hg38 by CrossMap. ChIP-seq: K562 ChIP-seq data were downloaded from ENCODE in genome build hg38. They were further organized into the following three categories: transcription factor binding sites (TFBS) and narrow and broad histone modifications (H3K27ac, H3K4me3, H3K9ac, H3K4me2, H2AFZ; H3K4me1, H3K27me3, H3K36me3, H3K9me3, H3K79me2, H3K9me1). Only replicated peaks were used for histone modifications. Hi-C: K562 Hi-C data were downloaded from GSE63535 (Rao et al., 2014) in genome build hg19. To compare it with hg38 while minimizing potential artifacts caused by upscaling Hi-C data, CHeX-seq from hg38 was upscaling to hg19 using CrossMap.Enhancers and super-enhancers: Experimentally validated human and mouse enhancers were downloaded from the VISTA database (Visel et al., 2007); super-enhancer data were downloaded from dbSUPER (Khan et al., 2016); DNA replication origins: K562 DNA replication origin data were downloaded from GEO (accession number GSE46189) in BED format using peaks pre-called by the authors. The original genome build was hg19, which was converted to hg38 by CrossMap. Enhancer / promoter interactions: In the UCSC genome browser, enhancers, promoters, and regulatory interactions were loaded from the database GeneHancer v 4.11 (Fisilevich et al., 2017) using only high-confidence ("double elite") data.

[0699] Example 4—Benchmarking CHeX-seq in human K562 cells

[0700] HK562 cells were chosen to benchmark the CHeX-seq procedure because this cell line has been selected for extensive analysis by ENCODE (ENCODE Project Consortium, 2012). After fixation, K562 cells were gravity deposited on poly-L-lysine-coated coverslips, then permeabilized and washed with PBS. Annealing of CHeX-seq fluorescently labeled primers to chemically fixed cells showed that the probes were concentrated in the nuclei of the cells ( Fig. 13B ). CHeX-seq primers were activated by irradiation with a 405 nm (UV) laser at 60% power and 30 μs per pixel, whereby a 45-80% decrease in fluorescence was observed ( Fig. 13B (Inset). This decrease is due to the loss of the fluorescent moiety and the release of the 3′-hydroxyl group to initiate DNA synthesis.

[0701] CHeX-seq reads were first preprocessed by a custom SCAP-T next-generation sequencing pipeline (available at github.com / safisher / ngs) and then mapped back to the UCSC hg38 (human) or UCSC mm10 (mouse) genome. Finally, additional QC procedures were applied to filter high-quality reads. The percentage of CHeX-seq reads mapping to different regions of the gene model was assessed along the length of the gene and its flanking regions, starting from the 5′ promoter region of all genes, through the transcription start site (TSS), 5′ untranslated region (UTR), exons and introns, 3′ UTR, and 3′ proximal region. Fig. 13C). K562 cells showed the highest proportion of CHeX-seq reads in intergenic regions (>50%), followed by introns (~30%), and then proximal promoters less than 1 kb from the transcription start site (TSS) (~6%). The promoter-proximal regions of genes (<1 kb) had more than 3 times more reads than distal regions (4-5 kb), consistent with open chromatin near the TSS. More specifically, TSS enrichment was observed in most single-cell samples, with weak or no enrichment in negative controls. Combining the signals from all non-control samples showed a clear peak centered on the TSS ( Fig.13D ), very similar to the TSS peak observed in ATAC-seq or DNase-seq assays (Buenrostro et al., 2013; Boyle et al., 2008). ATAC-seq data show a peak of sequencing reads around the TSS, while CHeX-seq data have a similar peak with a slightly extended slope following the peak in the 5′ to 3′ direction. Fig.13E Intracellular CHeX-seq signals from individual cells are shown, which are pooled for annotated features. These data suggest that chromatin tends to open near the start of a CDS (coding sequence), with a higher density of CHeX reads at the start of the CDS and a lower density of CheX reads within the CDS. This may be due to the high GC content observed in CDS regions, which can maintain the double-stranded state of DNA within open genomic regions. It may also reflect the dynamics of single-strand opening during transcriptional activity.

[0702] To assess how many K562 CHeX-seq sites correspond to expressed mRNAs, the CHeX-seq data were compared to a published K562 transcriptome dataset ( Fig.13F). These data indicate that approximately 64% of the transcriptome (15,688 genes) have CHeX-seq sites that correspond to expressed transcriptomes. Even with this relatively large overlap, there are still 7,286 CHeX-seq gene regions (approximately 32% of CHeX-seq genes) for which there is no evidence of transcription in the public transcriptome data. When comparing the CHeX-seq data to GRO-seq transcripts (a real-time transcription runoff assay (Danko et al., 2015; Lladser et al., 2017)), there is a similar number of overlapping genes (approximately 66%), while showing a reduction in CHeX-seq unique genes. Because the GRO-seq data is not significantly affected by RNA stability, it more accurately reflects genes that are actively transcribed from open chromatin regions. When assessing the gene ontology (GO) of K562 cell mRNA enriched for CHeX-seq sites, cell signaling, cell cycle, and GTPase regulation pathways were identified ( Figure 13G , the first 20 are shown). These data are consistent with the fact that K562 cells are a transformed cell line in which these pathways are functional.

[0703] The genome coverage of the CHeX-seq data is presented in the UCSC genome browser (Kent et al., 2002) compared to other open chromatin and epigenomic assays ( FIG. 14A to FIG. 14B ). For example, the OTUD5 gene region shows the mapping of CHeX-seq reads to three open chromatin assays (ATAC-seq, DNase-seq, FAIL-seq), highlighting open chromatin regions that overlap with each assay as well as regions that are unique to the assay ( Fig.14A ). 32 cells were mapped for the CHeX-seq sample, compared to a total of over 200 cells mapped for the other assays. In this particular view of the gene OTUD5, a regulatory interaction between the promoter of OTUD5 and one of its 3′ introns is noted, which is shared by all four open chromatin assays (purple rectangles). Due to the biological nature of the signal detected by each technology and due to the different chemistries of the assays, the different epigenomic assays have different genomic scales. To assess the relationship between the different epigenomic assays, the signal consistency of the 27 different assays in two different window sizes was calculated and the results were clustered ( Fig. 14B). Within the size scale of 10 kb windows, open chromatin assays (FAIRE-seq, DNase-seq, and ATAC-seq) clustered together, while CHeX-seq was in the same cluster but at a greater distance; this cluster also included histone methylation assays and replication origin assays. At a window size of 50 kb, CHeX-seq, ATAC-seq, and DNase-seq assays formed a tight cluster, with FAIL-seq outside of this cluster. Since the average size of a human gene is approximately 42 kb and the functional transcribed chromatin unit is approximately 50 kb (Hegedus et al., 2018), these data suggest that the same open chromatin-associated genes are identified using each of these programs, but that single-stranded open chromatin CHeX-seq positions may be displaced relative to positions in the other programs. A direct overlap is not expected, as other programs have a target bias toward dsDNA (ATAC-seq and FAIL-seq) or are indiscriminate (DNase-seq) compared to the ssDNA requirement of CHeX-seq. The CHeX-seq signal may be more sparse due to the limited number of cells analyzed, but these data highlight that both dsDNA and ssDNA are present within open chromatin regions and complement each other in the open chromatin landscape of the cell.

[0704] To confirm the single-stranded nature of some of the loci predicted by CHeX-seq, single-molecule FISH was performed on the CheX-seq predicted K562 single-stranded open chromatin site on chromosome 1 (630737-633960). Fig. 20A ). In addition to CheX-seq identifying the open chromatin state of this gene region, ATAC-seq also predicted that it was open, while in contrast, DNase showed limited openness and FAIRE predicted that it was not open. Eight 20-mer oligonucleotide probes were synthesized to this target region of human chromosome 1. These probes were labeled with ATT0 590 fluorophore at the 5′ end. When performing FISH, 3 strong positive spots were usually observed in a single cell nucleus ( Fig. 20B ). This trisomic signal is due to the complex karyotype of K562 cells, in which some cells have three copies of chromosome 1 (Gribble et al., 2000).

[0705] Next, K562 CHeX-seq priming sites were stratified according to their distance to the TSS of the cognate gene, and this distance was compared to the expression level of RNA from the same gene (Figure 15). Using three different RNA sources—population RNA (GSE32213), GRO-seq RNA (GSE60454), and single-cell RNA (scRNA, GSE 90063)—it was seen that when CHeX-seq priming sites were closer to the TSS, the corresponding RNA was generally present in higher abundance (Szlachta et al., 2018). This pattern was found in human and mouse neurons and astrocytes, as well as in neurons localized in mouse slices ( Fig.21 ). These data suggest that the plasticity of single-stranded DNA within a gene is regulated: that is, as transcription moves along the length of the gene, 5′-open sites become less available for hybridization, perhaps due to re-annealing of single-stranded regions. In this model, detectable CHeX-seq priming sites have different half-lives, and those close to the TSS remain single-stranded for longer and correspond to high levels of transcription. Therefore, CHeX-seq priming closer to the TSS would be more predictive of highly transcribed genes. These data are not simply due to differences in RNA stabilization rates, but also because GRO-seq RNA is newly synthesized nuclear RNA. One model is that TSS-proximal single-stranded strand type is associated with gene expression, and its accessibility decays as transcription proceeds, while single-stranded accessibility in more distal regions may be associated with other conformational regulation of the DNA.

[0706] Since the RNA Pol2 transcription complex binds to template DNA and synthesizes RNA transcripts in the 5′ to 3′ direction by transcribing the antisense strand, we assessed whether CHeX-seq probes might preferentially bind to the potentially more accessible sense strand, thereby generating an excess of “antisense” reads (see Fig.16A Schematic diagram in ). Fig. 16B The ratio of antisense to sense reads for different annotated regions of the gene model is shown. The results show a higher bias in read ratios for transcribed regions compared to non-transcribed regions of the genome, with a slight increase in bias from 5′UTR towards 3′UTR. Interestingly, promoter regions showed an opposite bias towards sense strand CHeX-seq products ( Fig. 16B ). This may reflect that the antisense strand binds to proteins containing Pol2 when replicating the antisense template, making the sense strand more available for CHeX-seq primer binding (Wang et al., 2014; Louder et al., 2016). This opposite trend at promoters may be related to bidirectional promoter activity (Wei et al., 2011).

[0707] Example 5 - In situ mouse brain tissue sections and dispersed single neuron analysis

[0708] To identify open chromatin sites in individual neurons in situ in adult mouse brain tissue, CheX-seq was applied to fixed adult brain tissue sections (100 μm) that were immunofluorescently labeled with an antibody detecting neuronal microtubule-associated protein 2 (MAP2) while the neurons were in their natural environment. The CheX-seq probes were then annealed to single-stranded DNA in the tissue sections (see schematic for a schematic). Fig.17A ). Fig. 17B The CA1 region of the hippocampus is shown with immunofluorescence against MAP2 (green) and CHeX-seq probes (red). CHeX-seq probes were placed in individual nuclei ( Fig. 17B The CHeX-primed DNA was removed, amplified, and sequenced. In comparing the open chromatin CHeX-seq sites from sliced ​​neurons with the expressed transcriptome from single cells ( Fig.17D ), CHeX-seq sites overlapped 59% with expressed RNA, while 88% of the transcriptome overlapped with CHeX-seq reads. This left 41% of CHeX-seq sites undetectable in RNA, while only 13% of the transcriptome showed no CHeX-seq open chromatin sites. These data suggest that there is a large amount of single-stranded open chromatin in fixed tissue sections that is not represented in the expressed transcriptome and may correspond to genes poised for transcription, sites of DNA replication, or other types of DNA organization. CHeX-seq reads in tissue sections can be further broken down to show 69% overlap of exonic regions with the transcriptome and 65% overlap of intronic regions with the transcriptome. This overlap suggests that both the chromatin landscape and the transcriptome are closely associated in cells located in their natural microenvironment.

[0709] To assess the pattern of single-stranded open chromatin regions in dispersed neurons, single fixed cultured mouse neurons ( Fig. 17C Since adult mouse neurons cannot be cultured and hippocampal cells are difficult to culture, open chromatin sites were assessed in mouse neonatal cortical neurons cultured for two weeks during which time they develop into dendrites. Dispersed mouse cortical neurons have similar chromatin sites to K562 cells ( Fig.13D ) and other cell types ( FIG. 22A to FIG. 22F) observed similar TSS peaks in the chromatin matrix, suggesting that they display the same TSS-open chromatin conformation. However, fewer total CHeX-seq reads mapping to the expressed transcriptome were found in dispersed cortical neurons (5,312) compared to in situ hippocampal neurons (9,709). Fig.17D ). It was found that for in situ neurons, 88% of the transcriptome was mapped to CHeX-seq reads, while for dispersed neuronal transcriptomes, only 48% of the transcriptome was mapped to CHeX-seq reads. However, the percentage of CHeX-seq reads corresponding to transcribed RNA was 68% for dispersed cells, compared to 59% for in situ neurons. In general, a higher percentage of CHeX-seq-positive regions showed signs of transcription in dispersed cortical neurons compared to in situ hippocampal cells, while a significantly lower percentage of transcribed RNA showed signs of CHeX-seq in dispersed cultures compared to slices. Although it is difficult to discern the relative contributions of cell type (although cortical and hippocampal cell transcriptomes are very similar (Zeisel et al., 2018)) and cell age to these data, one potential explanation is that there are more non-transcription-associated open chromatin sites in neurons in brain slices than in dispersed neurons.

[0710] When comparing CHeX-seq data from dispersed mouse cortical neurons to the average transcriptome of individual cortical neurons, there was a large overlap of single-stranded DNA sites with expressed RNA ( Fig.17D , right panel, upper left corner). Of the 7,728 CHeX-seq positives, 69% overlapped with the transcriptome, leaving 31% of single-stranded sites with either baseline transcribed RNA or no detectable transcribed RNA. Along with these data, 11,071 distinct transcripts were identified in single cells, 48% were associated with single-stranded open chromatin genes. To assess the systematic aspects of this comparison, Gene Ontology (GO) enrichment was assessed in shared genes and in genes unique to either assay in dispersed neurons. There were 235 GO molecular function terms shared between the open chromatin analysis and the transcriptome that were enriched at a Benjamini-Hochberg (BH) adjusted p-value <0.1, while at the same significance, there were 40 GO molecular function terms in CHeX-seq unique genes and 107 GO molecular function terms in transcriptome unique genes. Among the shared pathways were pathways for chromatin binding (p-value: 2.0×10 -14 ), pathway for calmodulin binding (p value: 1.9×10 -10) and many pathways associated with neuronal function. The signs that these pathways were enriched in both open chromatin and transcriptome of neurons are not surprising, as they generate normal cellular functions as well as some specialized functions of neurons. CHeX-seq unique genes showed a high propensity for pathways corresponding to regulatory RNA transcription (RNA polymerase II core promoter, p value: 1.3 × 10 -4 ; Transcriptional activation activity, p value: 4.0×10 -3 ; Enhancer sequence-specific DNA binding, p value: 6.1×10 -4 ) and a high tendency for pathways corresponding to the regulation of oxidative status (oxidoreductase activity, p value: 3.2 × 10 -4 ; Heme binding, p value: 1.3×10 -4 ). These sites may need to be open even when there is little transcribed RNA, so that the cell is ready to rapidly transcribe these RNAs when needed. In contrast, pathways enriched for transcriptome-unique genes included several involved in translation regulation (structural components of the ribosome, p value: 4.0 × 10 -40 ; rRNA binding, p value: 1.8×10 -7 ; Translation initiation factor activity, p value: 6.5×10 -9 ) and protein fidelity (ubiquitin protein ligase binding, p value: 1.1×10 -6 ; Unfolded protein binding, p value: 9.5×10 -7 ; Ubiquitin binding, p value: 5.8×10 -6 ) pathways, all of which meet the cellular requirements for functional proteins. Interestingly, there are many significantly enriched single-stranded DNA pathways that may be involved in preparing chromatin for opening and transcription (single-stranded DNA binding, p value: 2.3×10 -5 ; Helicase activity, p value: 1.2×10 -3 ). The single-stranded DNA pathway was also present in both shared genes and genes unique to CHeX-seq, but to a much lesser extent.

[0711] Interestingly, in neurons in situ, there was a significant relationship between the level of RNA expression within this gene and the number of CHeX-seq primed sites ( Fig.17E ). These data suggest that the number of CHeX-seq priming sites in a gene is indicative of the amount of transcription from that gene, with more sites indicating more transcription. This relationship is somewhat surprising because the steady-state level of RNA depends on factors other than transcriptional activity, such as RNA stability. A single open site can be associated with high levels of expression ( Fig.17E, left panel, far left), but the number of such sites is much smaller. The high number of open chromatin single-stranded sites in highly expressed genes in cells in tissue sections may reflect higher levels of activity, where genes have more frequent bursts of transcriptional activity and the open chromatin single-stranded state remains for extended periods of time. This is consistent with data showing that the variability of gene expression decreases when there are more CHeX-seq priming sites ( Fig.17F ). These data suggest that average-scale expression variability may be inversely correlated with the extent of quantification and base pair span of single-stranded DNA regions. Therefore, CHeX-seq priming measures may be correlated with temporal constancy of transcription as well as overall production levels, which would reflect the higher metabolic demands of the cell and the need for RNA to be expressed at constant high levels.

[0712] Priming rates were examined in units of extended genic regions, defined as the entire transcribed region (5'UTR, exons, introns, 3'UTR) with an additional 5 kb both upstream and downstream. For each extended genic region, priming events from 28 cultured neuronal samples and 15 in situ hippocampal neuronal samples were pooled together and Fisher's exact test was performed for different proportions, given the total reads in each treatment. A total of 86 significantly different priming rates (i.e., single-stranded regions) were found in extended genic regions after correction for multiple testing (Benjamin-Hochberg adjusted p-value < 0.05); 50 genic regions had greater CHeX-seq priming rates in dispersed cortical neurons compared to in situ hippocampal neurons, and 36 genic regions had greater in situ priming rates compared to dispersed cultures.

[0713] The 50 gene regions with greater initiation rates in cortical neurons in dispersed cultures include a different set of gene functions. It appears that biology has shifted after dispersion, with dispersed cell genes showing more single-stranded strand patterns for GO annotated genes associated with ciliary function, membrane function, and nucleotide binding. Since many genes in these functional categories are involved in cell shape in yeast (Hayles et al., 2013), these data suggest that shape-changing genes may be activated when cells are dispersed. When these 50 gene regions were examined to correspond to single-cell transcriptomes from dispersed cells, two of the genes that showed higher read recovery in dispersed cells were ACOX3 (acyl-CoA oxidase 3, an enzyme that works in peroxisomes (Vanhooren et al., 1996)) and SUDS 3 (subunit of the HDAC1-dependent SIN3A co-repressor complex (Fleischer et al., 2003)). SUDS3 is thought to enhance the activity of HDAC1 by regulating chromatin structure, thereby inhibiting transcription. It is possible that SUDS3 protein increases in dispersed cells and would act to reduce the number of open chromatin sites upon dispersion. This may be particularly true for the large number of nontranscriptional CHeX-seq-accessible single-stranded open chromatin sites identified in sliced ​​hippocampal neurons.

[0714] The 36 gene regions with high priming rates in in situ hippocampal cells were concentrated on mitochondrial-encoded genes, with 27 of the 37 mitochondrial-encoded genes showing significant differences (Table 2A and Table 2B). Mitochondrial DNA has been noted in other open chromatin assays but has typically been removed for nuclear DNA analysis (Montefiori et al., 2017). Mitochondrial DNA is not organized into chromatin like nuclear DNA, but rather has a nucleoid structure (containing single-stranded DNA regions) that is dynamically regulated and transcribed (Marom et al., 2019; Kucej et al., 2008; Tomaska ​​et al., 2001). For these gene regions, neurons from fixed sections showed an average of 15.7 CHeX-seq priming events per gene per cell, ranging from 6.8 events / cell to 32.1 events / cell. In contrast to these values, for neurons from culture, an average of only 0.016 priming events per mitochondrial-encoded gene per cell was found. Since CHeX-seq priming is limited by the spacing of single-stranded regions, it is generally not expected to have a very large number of priming events per gene region, and these events are assumed to represent single-stranded DNA found in multiple mitochondria in a given cell. These results suggest that mitochondrial activity, mitochondrial replication, and / or gene transcription may be reduced in neurons in culture. There were four cells in the tissue sections (single-cell samples) that also had almost no CHeX-seq priming in these 27 mitochondrial gene regions, while showing strong signals from other gene regions, indicating that the state of mitochondrial DNA activity is heterogeneous between individual cells.

[0715]

[0716]

[0717]

[0718]

[0719]

[0720]

[0721]

[0722]

[0723] Example 6 - Promoter openness in mouse and human astrocytes

[0724] The assay was further performed on newborn mouse and adult human astrocytes cultured for two weeks for comparison with neurons of the same species and age ( Fig.23A ). Mapping CHeX-seq reads to annotated gene models revealed that astrocytes had a higher proportion of CHeX-seq reads in the promoter regions of genes compared to neurons from the same species ( Fig. 23B ). These data are consistent with earlier studies (Thurman et al., 2012) showing that the chromatin landscape of dividing cells (astrocytes) has more DNase I-sensitive open chromatin around the promoter regions of genes compared to terminally differentiated cells (neurons). This is particularly interesting because the cells spanned a long age span, with the mouse cells being newborn and the human cells coming from subjects in the 50-70 year range. As noted above, promoter-proximal CHeX-seq priming is more indicative of gene transcription.

[0725] Example 7 - Open chromatin landscape across the mouse genome

[0726] Because CHeX-seq provides a genome-wide view of single-stranded open chromatin, we tested whether there are differential chromosome accessibility differences between mouse neurons and astrocytes. Fig.18 The CHeX-seq read density for all chromosomes (rows) for each individual cell (columns) in the is plotted as a heatmap. Two things emerge when looking at these data: 1) different cell types show different densities of single-stranded open chromatin across chromosomes, and 2) within a cell type, there are groupings of cells that may correspond to subtypes of the parental cell type. Fig.18 , rightmost panel) have greater read density on chromosomes 1, 2, and 9 than do scattered cortical neurons or astrocytes. In addition, these in situ neurons have three subgroups, one of which shows lower read density on chromosome 9 (green rectangles highlight dendrogram groupings). Astrocytes can similarly be divided into at least three groups ( Fig.18 , leftmost panel), where one of the discriminating factors is the density of open chromatin on chromosome 11. Since cells from multiple animals are included in each grouping, the grouping is not due to a batch effect. These data highlight the ability to characterize cell types based on their open chromatin state. Why chromosome open chromatin landscapes differ between cells is unclear, but these data reflect the dynamics of the genome. Future studies will lead to more fine-grained detailed maps of single-stranded open chromatin DNA dynamics.

[0727] Example 8 - CHeX-seq and LT-TISA in Immunoidentified Cells in Fixed Tissue Sections

[0728] Tissue sections through mouse brain were used to perform CHeX-seq and LT-TISA procedures on cells in fixed sections. Three cell types were assessed, including neurons (all with MAP2 (Izant and McIntosh, 1980) staining, inhibitory neurons identified by GAD (Xu et al., 2010) staining, and cholinergic neurons identified by choline acetyltransferase (ChAT) (Houser et al., 1985) staining, glial cells identified by GFAP (Eng et al., 2000), and endothelial cells identified by immunostaining with endothelial barrier antigen (EBA) (Ghabriel et al., 2000). Antibodies against each of these antigens are readily available and have been shown to be useful for staining the outline and cytoplasm of their designated cell types. As Fig.11 As indicated, 11 sections were stained simultaneously with antibodies against two or more antigens and then subjected to CHeX-seq and / or LT-TISA oligonucleotide hybridization. Fig.11 As shown, three fluorophores are used (two for MAP2 and GFAP antibodies, and one for CHeX-seq oligonucleotides), so the fluorescence emission spectra of the different fluorophores are different so that they can be distinguished. Various optimizations will be performed, including different slice thicknesses, different fixatives and fixation times, incubation times for various chemical detectors, optimization of CHeX-seq and LT-TISA cDNA synthesis protocols, and optimization of optical washes so that multiple cells can be analyzed.

[0729] Some embodiments include analysis of chromatin landscape using CHeX-seq analysis in neurons and glia under control conditions followed by fluoxetine-treated cells. Pooled cells of a particular cell type are assessed first, followed by single cells, to assess changes in intercellular responsiveness. These same treatments are performed in the following manner: LT-TISA of cytoplasmic RNA is multiplexed with CHeX-seq so that both chromatin state and cytoplasmic RNA abundance will be determined. To this end, primary cell cultures are prepared from mice treated with fluoxetine for 14 days, which is the time required for clinical effects to become apparent. Single neurons from these primary cultures are subjected to CHeX-seq. Once CHeX-seq baseline data is generated, CHeX-seq is combined with LT-TISA cytoplasmic RNA detection to assess the correlation between RNA abundance and the open single-stranded chromatin state of treated cells.

[0730] Example 9 - Characterization of 3D chromatin niches around selected genes

[0731] In some embodiments, the time course of 3D chromatin changes around specific genes associated with depression and known to respond to fluoxetine can be assessed ( Fig.12 ). Enhancer elements and other genomic / chromatin regulatory regions that regulate these specific genes will be detectable because their effects are thought to be exerted by means of genomic organization that positions regulatory elements near the genes being regulated. Specifically, using basic knowledge of chromatin responsiveness to 14 days of fluoxetine treatment, specifically responsive genes were selected for more detailed analysis of the time course of 3D chromatin niche modifications around these genes. Total CHeX-seq highlights all open regions of chromatin, but it is impossible to locate which open regions are close to each other except through cis-gene positioning. Since chromosome packaging can locate genes from distant chromosomal regions (including different chromosomes near the gene of interest), it is important to define chromosomal regions near the gene of interest. In addition, monitoring chromatin niche dynamics over a period of time from the start of fluoxetine treatment to 2 weeks will detail how these important regulatory niches change over time. In this gene-specific iteration of CHeX-seq, fluorescent in situ hybridization is performed using fixed cells / tissues to detect the location of specific neuronal genes. This gene-specific FISH signal was used to target multiphoton activation of CHeX-seq oligonucleotides only at the genes focused on by FISH.

[0732] In one such set of experiments, a FISH probe was used that was directed to an open chromatin region near the TSS of the CREB gene, which others have shown to be regulated by antidepressant activity (Zhang et al., 2015; Duman, 2013). This probe would produce a fluorescent signal at the CREB TSS that could be imaged. Following CREB TSS FISH, the added CHeX-seq oligonucleotides would diffuse throughout the tissue and bind to open regions of cellular chromatin. To specifically assess the chromatin structure around the CREB gene TSS, CREB FISH was imaged and then two-photon illumination was used to locally activate the CHeX-seq oligonucleotides at the CREB TSS FISH fluorescent sites ( Figure 6). The activated CHeX-seq oligonucleotide near the CREB gene extends from its 3' hydroxyl group, allowing DNA synthesis at a single-stranded open chromatin site near the CREB TSS. This gene niche-specific CHeX-seq program will identify sequences corresponding to chromatin regions near the CREB FISH site, which will be candidates for genomic regulatory elements (including enhancers) of CREB gene transcription. This method can be extended to any genomic DNA site specified by in situ hybridization. In addition, it can be used for higher throughput data generation using optical cleaning. This assay can also be developed for other genetic loci associated with other antidepressant effects.

[0733] ***

[0734] According to the present disclosure, all methods disclosed and claimed herein can be carried out and performed without undue experimentation. Although the compositions and methods of the present invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that the steps or sequence of steps of the methods and methods described herein can be changed without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain chemically and physiologically related reagents can replace the reagents described herein, while at the same time the same or similar results will be obtained. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0735] References

[0736] The following references and any other references cited in this application are specifically incorporated herein by reference to the extent they provide exemplary procedural or other details supplementary to those set forth herein.

[0737] Barnes et al., "Crystal Structure of a Transcribing RNA Polymerase IIComplex Reveals a Complete Transcription Bubble," Mol. Cell, 59:258-269, 2015.

[0738] Berton et al., "Essential role of BDNF in the mesolimbic dopamine pathway in social defeat stress," Science, 311:864-868, 2006.

[0739] Bieberstein et al.,“First exon length controls active chromatinsignatures and transcription,”Cell Rep.,2:62-68,2012.

[0740] Bjursell et al.,“Long regions of single-stranded DNA in human cells,”Nature,280:420-423,1979.

[0741] Borrelli et al.,“Decoding the epigenetic language of neuronalplasticity,”Neuron,60:961-974,2008.

[0742] Boyle et al.,“High-resolution mapping and characterization of openchromatin across the genome,”Cell,132:311-322,2008.

[0743] Buchhalter&Dichter,“Electrophysiological comparison of pyramidal andstellate nonpyramidal neurons in dissociated cell culture of rathippocampus,”Brain Res.Bull.,26:333-338,1991.

[0744] Buenrostro et al.,“Transposition of native chromatin for fast andsensitive epigenomic profiling of open chromatin,DNA-binding proteins andnucleosome position,”Nat.Methods,10:1213-1218,2013.

[0745] Buenrostro et al.,“Single-cell chromatin accessibility revealsprinciples of regulatory variation,”Nature,523:486-490,2015.

[0746] Chen et al.,“Variant GADL1 and response to lithium therapy in bipolarI disorder,”N.Engl.J.Med.,370:119-128,2014.

[0747] Choi et al.,“Programmable in situ amplification for multiplexedimaging of mRNA expression,”Nat.Biotechnol.,28:1208-1212,2010.

[0748] Choi et al.,“Mapping a multiplexed zoo of mRNA expression,”Development,143:3632-3637,2016.

[0749] Clark et al.,“scNMT-seq enables joint profiling of chromatinaccessibility DNA methylation and transcription in single cells,”Nat.Commun.,9:781,2018.

[0750] Core et al.,“Analysis of nascent RNA identifies a unifiedarchitecture of initiation regions at mammalian promoters and enhancers,”Nat.Genet.,46:1311-1320,2014.

[0751] Cornelison&Wold,“Single-cell analysis of regulatory gene expressionin quiescent and activated mouse skeletal muscle satellite cells,”Developmental Biology,191:270-283,1997.

[0752] Costa et al.,“Epigenetic targets in GABAergic neurons to treatschizophrenia,”Adv.Pharmacol.,54:95-117,2006.

[0753] Crino et al.,“Embryonic neuronal markers in tuberous sclerosis:single-cell molecular pathology,”Proc.Natl.Acad.Sci.U.S.A.,93:14152-14157,1996.

[0754] Cusanovich et al.,“Multiplex single cell profiling of chromatinaccessibility by combinatorial cellular indexing,”Science,348:910-914,2015.

[0755] Danko et al.,“Identification of active transcriptional regulatoryelements from GRO-seq data,”Nat.Methods,12:433-438,2015.

[0756] Dekker et al.,“Exploring the three-dimensional organization ofgenomes:interpreting chromatin interaction data,”Nat.Rev.Genet.,14:390-403,2013.

[0757] de la Torre-Ubieta&Bonni,“Transcriptional regulation of neuronalpolarity and morphogenesis in the mammalian brain,”Neuron,72:22-40,2011.

[0758] de Wit&de Laat,“A decade of 3C technologies:insights into nuclearorganization,”Genes Dev.,26:11-24,2012.

[0759] Dueck et al.,“Deep sequencing reveals cell-type-specific patterns ofsingle-cell transcriptome variation,”Genome Biol.,16:122,2015.

[0760] Dueck et al.,“Variation is function:Are single cell differencesfunctionally important?:Testing the hypothesis that single cell variation isrequired for aggregate function,”Bioessays,38:172-180,2016.

[0761] Duman,“Remodeling chromatin and synapses in depression,”Nat.Med.,19:267,2013.

[0762] Eberwine et al.,“Complementary DNA synthesis in situ:methods andapplications,”Methods Enzymol.,216:80-100,1992.

[0763] Eberwine et al.,“Analysis of gene expression in single live neurons,”Proc.Natl.Acad.Sci.U.S.A.,89:3010-3014,1992.

[0764] Ellis et al.,“Transcriptome analysis of cortical tissue revealsshared sets of downregulated genes in autism and schizophrenia,”Transl.Psychiatry,6:e817,2016.

[0765] Eng et al.,“Glial fibrillary acidic protein:GFAP-thirty-one years(1969-2000),”Neurochem.Res.,25:1439-1451,2000.

[0766] Feng et al.,“Identifying ChIP-seq enrichment using MACS,”Nat.Protoc.,7:1728-1740,2012.

[0767] Fishilevich et al.,“GeneHancer:genome-wide integration of enhancersand target genes in GeneCards,”Database(Oxford),2017:bax028,2017.

[0768] Fleischer et al.,“Identification and characterization of three newcomponents of the mSin3A corepressor complex,”Mol.Cell Biol.,23:3456-3467,2003.

[0769] Frankle et al.,“The synaptic hypothesis of schizophrenia,”Neuron,39:205-216,2003.

[0770] Fullard et al.,“Open chromatin profiling of human postmortem braininfers functional roles for non-coding schizophrenia loci,”Hum.Mol.Genet.,26:1942-1951,2017.

[0771] Ghabriel et al.,“Immunological targeting of the endothelial barrierantigen(EBA)in vivo leads to opening of the blood-brain barrier,”Brain Res.,878:127-135,2000.

[0772] Gribble et al.,“Cytogenetics of the chronic myeloid leukemia-derivedcell line K562:karyotype clarification by multicolor fluorescence in situhybridization,comparative genomic hybridization,and locus-specificfluorescence in situ hybridization,”Cancer Genet.Cytogenet.,118:1-8,2000.

[0773] Guidotti et al.,“Epigenetic GABAergic targets in schizophrenia andbipolar disorder,”Neuropharmacology,60:1007-1016,2011.

[0774] Hashimshony et al.,“CEL-Seq:single-cell RNA-Seq by multiplexed linearamplification,”Cell Rep.,2:666-673,2012.

[0775] Hayles et al.,“A genome-wide resource of cell cycle and cell shapegenes of fission yeast,”Open Biol.,3:130053,2013.

[0776] Hegedus et al.,“Endogenous single-strand DNA breaks at RNA polymeraseII promoters in Saccharomyces cerevisiae,”Nucleic Acids Res.,46:10649-10668,2018.

[0777] Heinz et al.,“Simple combinations of lineage-determiningtranscription factors prime cis-regulatory elements required for macrophageand B cell identities,”Mol.Cell.,38:576-589,2010.

[0778] Heller et al.,“Locus-specific epigenetic remodeling controlsaddiction-and depression-related behaviors,”Nat.Neurosci.,17:1720-1727,2014.

[0779] Houser et al.,“Immunocytochemical localization of cholineacetyltransferase in rat cerebral cortex:a study of cholinergic neurons andsynapses,”J.Comp.Neurol.,234:17-34,1985.

[0780] Huang&Akbarian,“GAD1 mRNA expression and DNA methylation inprefrontal cortex of subjects with schizophrenia,”PLoS One,2:e809,2007.

[0781] Insel&Wang,“Rethinking mental illness,”JAMA,303:1970-1971,2010.

[0782] Islam et al.,“Characterization of the single-cell transcriptionallandscape by highly multiplex RNA-seq,”Genome Res.,21:1160-1167,2011.

[0783] Islam et al.,“Highly multiplexed and strand-specific single-cell RNA5′end sequencing,”Nat.Protoc.,7:813-828,2012.

[0784] Izant&McIntosh,“Microtubule-associated proteins:a monoclonal antibodyto MAP2 binds to differentiated neurons,”Proc.Natl.Acad.Sci.U.S.A.,77:4741-4745,1980.

[0785] Kent et al.,“The human genome browser at UCSC,”Genome Res.,12:996-1006,2002.

[0786] Khan&Zhang,“dbSUPER:a database of super-enhancers in mouse and humangenome,”Nucleic Acids Res.,44:D164-171,2016.

[0787] Kim&Eberwine,“RNA:state memory and mediator of cellular phenotype,”Trends Cell Biol.,20:311-318,2010.

[0788] Kolovos et al.,“Targeted Chromatin Capture(T2C):a novel highresolution high throughput method to detect genomic interactions andregulatory elements,”Epigenetics Chromatin,7:10,2014.

[0789] Kouzine et al.,“Permanganate / S1 Nuclease Footprinting Reveals Non-BDNA Structures with Regulatory Potential across a Mammalian Genome,”CellSyst.,4:344-356 e347,2017.

[0790] Kozlenkov et al.,“Differences in DNA methylation between humanneuronal and glial cells are concentrated in enhancers and non-CpG sites,”Nucleic Acids Res.,42:109-127,2014.

[0791] Kozlenkov et al.,“Substantial DNA methylation differences between twomajor neuronal subtypes in human brain,”Nucleic Acids Res.,44:2593-2612,2016.

[0792] Kucej et al.,“Mitochondrial nucleoids undergo remodeling in responseto metabolic cues,”J.Cell Sci.,121:1861-1868,2008.

[0793] Kumar et al.,“Chromatin remodeling is a key mechanism underlyingcocaine-induced plasticity in striatum,”Neuron,48:303-314,2005.

[0794] Lai et al.,“Integrator mediates the biogenesis of enhancer RNAs,”Nature,525:399-403,2015.

[0795] Lee et al.,“Fluorescent in situ sequencing(FISSEQ)of RNA for geneexpression profiling in intact cells and tissues,”Nat.Protoc.,10:442-458,2015.

[0796] Lladser et al.,“RNA Pol II transcription model and interpretation ofGRO-seq data,”J.Math Biol.,74:77-97,2017.

[0797] Louder et al.,“Structure of promoter-bound TFIID and model of humanpre-initiation complex assembly,”Nature,531:604-609,2016.

[0798] Marom et al.,“mtDNA Chromatin-like Organization Is GraduallyEstablished during Mammalian Embryogenesis,”iScience,12:141-151,2019.

[0799] Martinowich et al.,“DNA methylation-related chromatin remodeling inactivity-dependent BDNF gene regulation,”Science,302:890-893,2003.

[0800] Maurano et al.,“Systematic localization of common disease-associatedvariation in regulatory DNA,”Science,337:1190-1195,2012.

[0801] McCarthy et al.,“De novo mutations in schizophrenia implicatechromatin remodeling and support a genetic overlap with autism andintellectual disability,”Mol.Psychiatry,19:652-658,2014.

[0802] Miyashiro et al.,“On the nature and differential distribution ofmRNAs in hippocampal neurites:implications for neuronal functioning,”Proc.Natl.Acad.Sci.U.S.A.,91:10800-10804,1994.

[0803] Miyashiro et al.,“RNA cargoes associating with FMRP reveal deficitsin cellular functioning in Fmr1 null mice,”Neuron,37:417-431,2003.

[0804] Miyashiro&Eberwine,“Identification of RNA cargoes by antibody-positioned RNA amplification,”Cold Spring Harb.Protoc.,2015:434-441,2015.

[0805] Mo et al.,“Epigenomic Signatures of Neuronal Diversity in theMammalian Brain,”Neuron,86:1369-1384,2015.

[0806] Moffitt&Zhuang,“RNA Imaging with Multiplexed Error-RobustFluorescence In Situ Hybridization(MERFISH),”Methods Enzymol.,572:1-49,2016.

[0807] Montefiori et al.,“Reducing mitochondrial reads in ATAC-seq usingCRISPR / Cas9,”Sci.Rep.,7:2451,2017.

[0808] Rao et al.,“A 3D map of the human genome at kilobase resolutionreveals principles of chromatin looping,”Cell,159:1665-1680,2014.

[0809] Renthal et al.,“Histone deacetylase 5 epigenetically controlsbehavioral adaptations to chronic emotional stimuli,”Neuron,56:517-529,2007.

[0810] Rotem et al.,“Single-cell ChIP-seq reveals cell subpopulationsdefined by chromatin state,”Nat.Biotechnol.,33:1165-1172,2015.

[0811] Roussos et al.,“A role for noncoding variation in schizophrenia,”CellRep.,9:1417-1429,2014.

[0812] Ruzicka et al.,“Circuit-and Diagnosis-Specific DNA MethylationChanges at gamma-Aminobutyric Acid-Related Genes in Postmortem HumanHippocampus in Schizophrenia and Bipolar Disorder,”JAMA Psychiatry,72:541-551,2015.

[0813] SantaLucia&Hicks,“The thermodynamics of DNA structural motifs,”Annu.Rev.Biophys.Biomol.Struct.,33:415-440,2004.

[0814] Scheer et al.,“High sensitivity immunolocalization of double andsingle-stranded DNA by a monoclonal antibody,”Eur.J.Cell Biol.,43:358-371,1987.

[0815] Seifuddin et al.,“Systematic review of genome-wide gene expressionstudies of bipolar disorder,”BMC Psychiatry,13:213,2013.

[0816] Shah et al.,“seqFISH Accurately Detects Transcripts in Single Cellsand Reveals Robust Spatial Organization in the Hippocampus,”Neuron,94:752-758e1,2017.

[0817] Sharma et al.,“Valproic acid and chromatin remodeling inschizophrenia and bipolar disorder:preliminary results from a clinicalpopulation,”Schizophr.Res.,88:227-231,2006.

[0818] Sherwood et al.,“Discovery of directional and nondirectional pioneertranscription factors by modeling DNase profile magnitude and shape,”Nat.Biotechnol.,32:171-178,2014.

[0819] Simonis et al.,“Nuclear organization of active and inactive chromatindomains uncovered by chromosome conformation capture-on-chip(4C),”Nat.Genet.,38:1348-1354,2006.

[0820] Spaethling et al.,“Primary Cell Culture of Live NeurosurgicallyResected Aged Adult Human Brain Cells and Single Cell Transcriptomics,”CellRep.,18:791-803,2017.

[0821] Sun et al.,“ACF chromatin-remodeling complex mediates stress-induceddepressive-like behavior,”Nat.Med.,21:1146-1153,2015.

[0822] Szlachta et al.,“Alternative DNA secondary structure formationaffects RNA polymerase II promoter-proximal pausing in human,”Genome Biol.,19:89,2018.

[0823] Tay et al.,“Single-cell NF-kappaB dynamics reveal digital activationand analogue information processing,”Nature,466:267-271,2010.

[0824] Tecott et al.,“In situ transcription:specific synthesis ofcomplementary DNA in fixed tissue sections,”Science,240:1661-1664,1988.

[0825] Thurman et al.,“The accessible chromatin landscape of the humangenome,”Nature,489:75-82,2012.

[0826] Tomaska et al.,“Mitochondrial single-stranded DNA-binding proteins:insearch for new functions,”Biol.Chem.,382:179-186,2001.

[0827] Tsankova et al.,“Epigenetic regulation in psychiatric disorders,”Nat.Rev.Neurosci.,8:355-367,2007.

[0828] Van Gelder et al.,“Amplified RNA synthesized from limited quantitiesof heterogeneous cDNA,”Proc.Natl.Acad.Sci.U.S.A.,87:1663-1667,1990.

[0829] Vanhooren et al.,“Mammalian peroxisomal acyl-CoA oxidases.I.Molecularcharacterization of rat pristanoyl-CoA oxidase,”Ann.N.Y.Acad.Sci.,804:674-675,1996.

[0830] Varodayan&Harrison,“HSF1 transcriptional activity mediates alcoholinduction of Vamp2 expression and GABA release,”Front.Integr.Neurosci.,7:89,2013.

[0831] Vasquez et al.,“Manipulating the mammalian genome by homologousrecombination,”Proc.Natl.Acad.Sci.U.S.A.,98:8403-8410,2001.

[0832] Vialou et al.,“Epigenetic mechanisms of depression and antidepressantaction,”Annu.Rev.Pharmacol.Toxicol.,53:59-87,2013.

[0833] Visel et al.,“VISTA Enhancer Browser--a database of tissue-specifichuman enhancers,”Nucleic Acids Res.,35:D88-92,2007.

[0834] Wang et al.,“A source of the single-stranded DNA substrate foractivation-induced deaminase during somatic hypermutation,”Nat.Commun.,5:4137,2014.

[0835] Wei et al.,“Functional consequences of bidirectional promoters,”Trends Genet.,27:267-276,2011.

[0836] Wu et al.,“Quantitative assessment of single-cell RNA-sequencingmethods,”Nat.Methods,11:41-46,2014.

[0837] Xiao et al.,“The DNA methylome and transcriptome of different brainregions in schizophrenia and bipolar disorder,”PLoS One,9:e95875,2014.

[0838] Xu et al.,“Immunochemical characterization of inhibitory mousecortical neurons:three chemically distinct classes of inhibitory cells,”J.Comp.Neurol.,518:389-404,2010.

[0839] Yu et al.,“Chromatin dynamics during the differentiation of long-termhematopoietic stem cells to multipotent progenitors,”Blood Adv.,1:887-898,2017.

[0840] Zeisel et al., “Molecular Architecture of the Mouse Nervous System,” Cell, 174:999-1014 e1022, 2018.

[0841] Zhang et al., “Increased Variability of Genomic Transcription in Schizophrenia,” Sci.Rep., 5:17995, 2015.

[0842] Zhao et al., “CrossMap: a versatile tool for coordinate conversion between genome assemblies,” Bioinformatics, 30:1006-1007, 2014.

[0843] Zhou & Paull, “Direct measurement of single-stranded DNA intermediates in mammalian cells by quantitative polymerase chain reaction,” Anal Biochem., 479:48-50, 2015.

[0844] Zhu et al., “Genome-wide chromatin state transitions associated with developmental and environmental cues,” Cell, 152:642-654, 2013.

[0845] Ziller et al., “Dissecting neural differentiation regulatory networks through epigenetic footprinting,” Nature, 518:355-359, 2015. Sequence Listing <110> Agilent Technologies, Inc. The Trustees Of The University of Pennsylvania <120> Compositions and methods for chromatin and gene expression analysis in single cells <130> 20190021-04 (AGLT.P0014WO) <140> Not yet assigned <141> 2020-01-07 <150> US 62 / 898,824 <151> 2019-09-11 <150> US 62 / 789,073 <151> 2019-01-07 <160> 19 <170> PatentIn Version 3.5 <210> 1 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <220> <221> N_Area <222> (45)..(59) <223> n is any nucleotide <220> <221> misc_features <222> (45)..(59) <223> n is a, c, g, or t <400> 1 ggagaattgt aatacgactc actataggga gacgcgtgat cacgnnnnnnnnnnnnnnnnt 60 gtg 63 <210> 2 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <220> <221> N_Area <222> (45)..(59) <223> n is any nucleotide <220> <221> misc_features <222> (45)..(59) <223> n is a, c, g, or t <400> 2 ggagaattgt aatacgactc actataggga gacgcgtgat cacgnnnnnnnnnnnnnnnnt 60 <210> 3 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <220> <221> N_Area <222> (4)..(18) <223> n is any nucleotide <220> <221> misc_features <222> (4)..(18) <223> n is a, c, g, or t <400> 3 aaannnnnnnnnnnnnnnncg tgatcacgcg tctccctata gtgagtcgta ttacaattct 60 cc 62 <210> 4 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 4 ggagaattgt aatacgactc actataggga gacgcgtgat cacgaca 47 <210> 5 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 5 cgtgatcacg cgtctcccta tagtgagtcg tattacaatt ctcc 44 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <220> <221> N_Area <222> (8)..(22) <223> n is any nucleotide <220> <221> misc_features <222> (8)..(22) <223> n is a, c, g, or t <400> 6 atgaggannn nnnnnnnnnn nnt 23 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <220> <221> N_Area <222> (4)..(18) <223> n is any nucleotide <220> <221> misc_features <222> (4)..(18) <223> n is a, c, g, or t <400> 7 aaannnnnnn nnnnnnnntc ctcattgt 28 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 8 tagggagacg cgtgatca 18 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 9 tagggagacg cgtgagtt 18 <210> 10 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 10 gcgccattga ccaggatttt cccccccccc cccc 34 <210> 11 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 11 gcgccattga ccaggatttt c 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 12 tccttagctg ttgcagaaat 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 13 cgttcagttg atgcagagtg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 14 agaagcagct tcaaacctgc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 15 aagttagctt tacagtgggc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 16 ggtgttggtt ctctttaatct 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 17 atttagttgg ggcatttcac 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 18 ttatggtggg tcatacggta 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 19 tagtgtaagg agtatggggg 20

Claims

1. An oligonucleotide molecule, comprising from 5' to 3' an amplification segment, a spacer segment, an index barcode segment, a hybridization segment and a reversible termination nucleotide, wherein the reversible termination nucleotide is a photoactivatable termination nucleotide, the amplification segment is an RNA polymerase promoter, the hybridization segment comprises a random nucleotide sequence or a known nucleotide sequence, the reversible termination nucleotide comprises a nitrobenzyl group and a fluorescent label, and the photoactivatable termination nucleotide comprises a structure of the following formula: in: R1 is O or S; R2 is hydrogen or hydroxyl; R3 is an alkyl group (C≤8) or substituted alkyl (C≤8) ; R4 is hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol; alkyl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups; R5, R6 and R7 are each independently: hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol; alkyl (C≤6) , alkenyl (C≤6) , alkynyl (C≤6) , aromatic (C≤6) , arylalkyl (C≤8) , heteroaryl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups; A set of the following formulas: in X is -O-, -S-, or -NH-; or Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , or a substituted form of any of these groups; Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ; n is an integer from 0 to 6; and m is an integer from 0 to 6; or - linker-reporter; or its tautomers or optical isomers.

2. The oligonucleotide molecule according to claim 1, wherein the amplification segment comprises 7 to 50 nucleotides.

3. The oligonucleotide molecule of claim 1, wherein the known nucleotide sequence is complementary to a target genomic or mitochondrial DNA sequence. The oligonucleotide molecule according to claim 1 , wherein the known nucleotide sequence is complementary to a target RNA sequence.

5. The oligonucleotide molecule according to any one of claims 1 to 3, wherein the hybridizing segment comprises a poly-T sequence.

6. The oligonucleotide molecule according to any one of claims 1 to 3, wherein the hybridizing segment comprises 7 to 30 nucleotides.

7. The oligonucleotide molecule according to any one of claims 1 to 3, wherein the hybridizing segment comprises 15 nucleotides. The oligonucleotide molecule according to claim 1 , wherein R 7 is methoxy.

9. The oligonucleotide molecule of claim 8, wherein the photoactivatable terminator nucleotide comprises a structure of the formula: Wherein R5 is -linker-reporter.

10. The oligonucleotide molecule of claim 9, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

11. A population of oligonucleotide molecules, each oligonucleotide molecule comprising, from 5' to 3', an amplification segment, a spacer segment, an index barcode segment, a hybridization segment, and a reversible termination nucleotide, wherein the reversible termination nucleotide is a photoactivatable termination nucleotide, the amplification segment is an RNA polymerase promoter, the hybridization segment comprises a degenerate nucleotide sequence or one or more known nucleotide sequences, the reversible termination nucleotide comprises a nitrobenzyl group and a fluorescent label, and the photoactivatable termination nucleotide comprises a structure of the formula: in: R1 is O or S; R2 is hydrogen or hydroxyl; R3 is an alkyl group (C≤8) or substituted alkyl (C≤8) ; R4 is hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol; alkyl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups; R5, R6 and R7 are each independently: hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol; alkyl (C≤6) , alkenyl (C≤6) , alkynyl (C≤6) , aromatic (C≤6) , arylalkyl (C≤8) , heteroaryl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups; A set of the following formulas: in X is -O-, -S-, or -NH-; or Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , or a substituted form of any of these groups; Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ; n is an integer from 0 to 6; and m is an integer from 0 to 6; or - linker-reporter; or its tautomers or optical isomers.

12. The population of claim 11, wherein the amplified segment comprises 7 to 50 nucleotides.

13. The population of claim 11, wherein each nucleic acid molecule in the population comprises a unique hybridization segment sequence.

14. The population of claim 11, wherein each known nucleotide sequence is complementary to a target genomic or mitochondrial DNA sequence.

15. The population of claim 14, wherein each known nucleotide sequence is complementary to a target RNA sequence.

16. A population according to any one of claims 11-15, wherein the hybridization segment comprises a poly-T sequence.

17. A population according to any one of claims 11-15, wherein the hybridization segment comprises 7 to 30 nucleotides.

18. A population according to any one of claims 11-15, wherein the hybridization segment comprises 15 nucleotides.

19. A population according to claim 11, wherein R7 is methoxy.

20. The population of claim 19, wherein the photoactivatable terminator nucleotide comprises a structure of the formula: Wherein R5 is -linker-reporter.

21. The population of claim 20, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

22. A method for identifying open DNA regions in a cell, the method comprising: (a) introducing a population of oligonucleotide molecules into the cell, wherein each molecule comprises, from 5' to 3', an amplification segment, a spacer segment, an index barcode segment, a hybridization segment, and a reversible terminator nucleotide, wherein the reversible terminator nucleotide is a photoactivatable terminator nucleotide, the amplification segment is an RNA polymerase promoter, the hybridization segment comprises a degenerate nucleotide sequence or one or more known nucleotide sequences, the reversible terminator nucleotide comprises a nitrobenzyl group and a fluorescent label, and the photoactivatable terminator nucleotide comprises a structure of the formula: in: R1 is O or S; R2 is hydrogen or hydroxyl; R3 is an alkyl group (C≤8) or substituted alkyl (C≤8) ; R4 is hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol; alkyl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups; R5, R6 and R7 are each independently: hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol; alkyl (C≤6) , alkenyl (C≤6) , alkynyl (C≤6) , aromatic (C≤6) , arylalkyl (C≤8) , heteroaryl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups; A set of the following formulas: in X is -O-, -S-, or -NH-; or Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , or a substituted form of any of these groups; Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ; n is an integer from 0 to 6; and m is an integer from 0 to 6; or - linker-reporter; or a tautomer or an optical isomer thereof; (b) incubating the cells under conditions that allow annealing of the hybridized segments of the population of oligonucleotide molecules to open DNA regions; (c) activating at least a portion of the annealed oligonucleotide molecules to expose extendable 3' hydroxyl groups; and (d) synthesizing cDNA from the open DNA by extending the activated oligonucleotide molecule from its extendable 3' hydroxyl group.

23. The method of claim 22, wherein the amplified segment comprises 7 to 50 nucleotides.

24. The method of claim 22, wherein each nucleic acid molecule in the population comprises a unique hybridization segment sequence.

25. The method of claim 22, wherein each known nucleotide sequence is complementary to a target genomic or mitochondrial DNA sequence.

26. The method of any one of claims 22-25, wherein the hybridization segment comprises 7 to 30 nucleotides.

27. The method of any one of claims 22-25, wherein the hybridization segment comprises 15 nucleotides.

28. The method of claim 22, wherein R7 is methoxy.

29. The method of claim 28, wherein the photoactivatable terminator nucleotide comprises a structure of the formula: Wherein R5 is -linker-reporter.

30. The method of claim 29, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

31. The method according to any one of claims 22-25, wherein the population of oligonucleotide molecules introduced in process (a) is a population of oligonucleotide molecules according to any one of claims 12-15 and 18-23.

32. The method of any one of claims 22-25, wherein the cells are fixed.

33. The method of any one of claims 22-25, wherein the open DNA is open chromatin, and wherein activation comprises exposing the nucleus to ultraviolet light.

34. The method of any one of claims 22-25, wherein activation is performed throughout the cell nucleus.

35. The method of any one of claims 22-25, wherein the open DNA is open mitochondrial DNA, wherein activation comprises exposing at least one mitochondria in the cell to ultraviolet light.

36. The method of any one of claims 22-25, wherein activation is performed on more than one mitochondria in the cell.

37. The method of any one of claims 22-25, wherein activation is performed at a transcription start site within the nucleus or mitochondria.

38. The method of claim 37, wherein the transcription start site is identified based on the location of the gene of interest.

39. The method of claim 38, wherein in situ hybridization is used to locate the transcription start site.

40. The method of claim 39, wherein activation at the transcription start site comprises exposing the transcription start site to multi-photon excitation based on an in situ hybridization signal.

41. The method of any one of claims 22-25, wherein synthesizing cDNA comprises adding a DNA-dependent DNA polymerase.

42. The method of any one of claims 22-25, further comprising processing the synthesized cDNA to produce double-stranded cDNA comprising the index barcode segment and the amplification segment of the oligonucleotide.

43. The method of claim 42, further comprising amplifying the double-stranded cDNA.

44. The method of claim 43, wherein amplification comprises PCR, rolling circle amplification, or RNA amplification.

45. The method of claim 43, further comprising obtaining the sequence of at least a portion of the double-stranded cDNA.

46. ​​The method of claim 45, further comprising aligning the sequence with a genomic or mitochondrial sequence to identify open DNA regions.

47. The method of any one of claims 22-25, wherein the method is a multiplex method, wherein the method is performed sequentially on two or more cells in a sample.

48. The method of any one of claims 22-25, wherein the method is a multiplex method, wherein the method is performed sequentially for two or more transcription start sites in the cell.

49. The method of claim 48, wherein the population of oligonucleotide molecules introduced during each round of multiplex amplification comprises a unique index barcode segment.

50. The method of any one of claims 22-25, wherein the method is further defined as a method for classifying cells.

51. The method of any one of claims 22-25, wherein the method is further defined as a method for predicting or determining a subtype of a cell.

52. A method for identifying RNA expressed in a cell, the method comprising: (a) introducing a population of oligonucleotide molecules into the cell, wherein each molecule comprises, from 5' to 3', an amplification segment, a spacer segment, an index barcode segment, a hybridization segment, and a reversible terminator nucleotide, wherein the reversible terminator nucleotide is a photoactivatable terminator nucleotide, the amplification segment is an RNA polymerase promoter, the hybridization segment comprises one or more known nucleotide sequences, the reversible terminator nucleotide comprises a nitrobenzyl group and a fluorescent label, and the photoactivatable terminator nucleotide comprises a structure of the formula: in: R1 is O or S; R2 is hydrogen or hydroxyl; R3 is an alkyl group (C≤8) or substituted alkyl (C≤8) ; R4 is hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol; alkyl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups; R5, R6 and R7 are each independently: hydrogen, hydroxy, halide, amino, nitro, cyano, azido or thiol; alkyl (C≤6) , alkenyl (C≤6) , alkynyl (C≤6) , aromatic (C≤6) , arylalkyl (C≤8) , heteroaryl (C≤6) , acyl (C≤6) , alkoxy (C≤6) , acyloxy (C≤6) , alkylamino (C≤6) , dialkylamino (C≤6) , amide (C≤6) , or a substituted form of any of these groups; A set of the following formulas: in X is -O-, -S-, or -NH-; or Alkanediyl (C≤12) , alkene diyl (C≤12) , acetylene (C≤12) , or a substituted form of any of these groups; Y is -O-, -NH-, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ; n is an integer from 0 to 6; and m is an integer from 0 to 6; or - linker-reporter; or a tautomer or an optical isomer thereof; (b) incubating the cell under conditions that allow annealing of the hybridizing segments of the population of oligonucleotide molecules to the expressed RNA; (c) activating at least a portion of the annealed oligonucleotide molecules to expose extendable 3' hydroxyl groups; and (d) synthesizing cDNA from the expressed RNA by extending the activated oligonucleotide molecule from its extendable 3' hydroxyl group.

53. The method of claim 52, wherein the amplified segment comprises 7 to 50 nucleotides.

54. The method of claim 52, wherein the hybridization segment comprises a poly-T sequence.

55. The method of claim 52, wherein each known nucleotide sequence is complementary to a target RNA sequence.

56. The method of any one of claims 52-55, wherein the hybridization segment comprises 7 to 30 nucleotides.

57. The method of any one of claims 52-55, wherein the hybridization segment comprises 15 nucleotides.

58. The method of claim 52, wherein R7 is methoxy.

59. The method of claim 58, wherein the photoactivatable terminator nucleotide comprises a structure of the formula: Wherein R5 is -linker-reporter.

60. The method of claim 59, wherein the photoactivatable terminator nucleotide comprises a structure of the formula:

61. The method according to any one of claims 52-55, wherein the population of oligonucleotide molecules introduced in process (a) is a population of oligonucleotide molecules according to any one of claims 11-13 and 15-21.

62. The method of any one of claims 52-55, wherein the cells are fixed.

63. The method of any one of claims 52-55, wherein activation comprises exposing the cytoplasm to ultraviolet light.

64. The method of any one of claims 52-55, wherein activation is performed throughout the cytoplasm.

65. The method of any one of claims 52-55, wherein activation is performed at an axonal or dendritic site within the cytoplasm.

66. The method of any one of claims 52-55, wherein synthesizing cDNA comprises adding an RNA-dependent DNA polymerase.

67. The method of any one of claims 52-55, further comprising processing the synthesized cDNA to produce double-stranded cDNA, the double-stranded cDNA comprising the index barcode segment and the amplification segment of the oligonucleotide.

68. The method of claim 67, further comprising amplifying the double-stranded cDNA.

69. The method of claim 68, wherein amplification comprises PCR, rolling circle amplification, or RNA amplification.

70. The method of claim 68, further comprising obtaining the sequence of at least a portion of the double-stranded cDNA, thereby identifying the expressed RNA.

71. The method of any one of claims 52-55, wherein the method is a multiplex method, wherein the method is performed sequentially on two or more axonal or dendritic sites in the cell.

72. The method of any one of claims 52-55, wherein the method is a multiplex method, wherein the method is performed sequentially on two or more cells in a sample.

73. The method of claim 72, wherein the population of oligonucleotide molecules introduced during each round of multiplex amplification comprises a unique index barcode segment.

74. A kit comprising a population of oligonucleotide molecules according to any one of claims 11-21, and at least one of a DNA polymerase, an RNA polymerase, a reverse transcriptase, a deoxyribonucleotide, and a ribonucleotide.

Images