Methods and systems for parallel spatially resolved single-cell profiling

Abstract

Provided herein are, inter alia, are systems and methods for detecting the transcriptional activity of an enhancer. In addition, provided herein are, inter alia, are systems and methods for use in parallel spatially resolved single-cell profiling. Also provided herein are, inter alia, compositions and kits for use in methods of detecting the transcriptional activity of an enhancer.

Description

METHODS AND SYSTEMS FOR PARALLEL SPATIALLY RESOLVED SINGLE¬CELL PROFILINGRELATED APPLICATION DATA

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Patent Application No. 63 / 652,580, filed on May 28, 2024, which is hereby incorporated by reference in its entirety and for all purposes.SEQUENCE LISTING

[0002] The material in the accompanying Sequence Listing is hereby incorporated by reference in its entirety. The accompanying sequence listing file, named “048537- 667001WO_SL_ST26.xml” was created on May 28, 2025 and is 427.322 bytes in size.BACKGROUND

[0003] Whole genome sequencing has become routine with the advancement of next-gen DNA sequencing technologies, but how the genome sequence encodes the complex cis regulatory code is still not clear. Deciphering this code is pivotal for understanding the molecular underpinnings of human diseases, such as cardiovascular disease, diabetes, and Alzheimer’s disease, conditions that have been linked to non-coding germline mutations suspected to disrupt gene regulation. The challenge in elucidating the cis-regulatory code stems from the cell-specific and context- dependent interactions between transcription factors (TFs) and the cis regulatory elements such as enhancers, promoters, and insulator elements, which current methodologies struggle to accurately capture. Provided herein, inter alia, are systems and methods to address these and other problems in the art.BRIEF SUMMARY

[0004] In an aspect is provided a method of detecting transcriptional activity of an enhancer sequence in a cell, the method including: (a) delivering a plasmid to a cell, wherein the plasmid includes a DNA sequence including a protein encoding sequence, an enhancer sequence and a barcode sequence associated with the enhancer sequence and allowing the cell to transcribe the plasmid thereby producing a barcoded RNA transcript including the protein encoding sequence, the enhancer sequence, and the barcode sequence, wherein the barcode sequence is operablylinked to the enhancer sequence; (b) fixing the cell; (c) contacting the barcoded RNA transcript with a plurality of different encoding probes and allowing each of the plurality of different encoding probes to hybridize to a different subsequence within the barcoded RNA transcript thereby forming a plurality of encoding probe RNA transcript hybridized complexes; (d) contacting the plurality of encoding probe RNA transcript hybridized complexes with a first plurality of different adapter probes and allowing each of the different adapter probe in the first plurality of different adapter probes to hybridize to a different encoding probe bound to the barcoded RNA transcript thereby forming a first plurality of adapter probe RNA transcript hybridized complexes; (e) contacting the first plurality of adapter probe RNA transcript hybridized complexes with a plurality of different fluorescent readout probes and allowing each of the different fluorescent readout probes to hybridize to a different adapter probe bound to the barcoded RNA transcript thereby forming a first plurality of fluorescent readout probe RNA transcript hybridized complexes; and (f) detecting the first plurality of different fluorescent readout probes hybridized to the barcoded RNA transcript, thereby detecting the barcoded RNA transcript and the enhancer transcriptional activity.

[0005] In another aspect is provided an expression vector including a DNA sequence encoding a protein encoding sequence, an enhancer sequence, and a barcode sequence.

[0006] In another aspect is provided a kit including the expression vector provided herein including embodiments thereof.

[0007] In another aspect is provided a method of quantifying transcriptional activity in a cell, the method including: (i) transfecting the cell with a reporter construct; (ii) allowing the cell to express the reporter construct; (iii) contacting the cell with a probe; (iv) quantifying expression of the reporter construct, thereby quantifying transcriptional activity’ in the cell.

[0008] In another aspect is provided a method of quantify ing cis-regulatory element activity7in a cell, the method including: (i) transfecting the cell with a reporter construct;(ii) allowing the cell to express the reporter construct: (iii) contacting the cell with a probe; (iv) quantifying expression of the reporter construct, thereby quantifying cis-regulatory element activity’ in the cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIGS. 1A-1C show STARR-FISH (self-transcribing active regulatory region coupled with fluorescence in situ hybridization) enables quantitative measurements of enhancer activity at both single cell and spatial resolution. FIG. 1A: Schematic overview of STARR-FISH. Plasmid libraries containing enhancers of interest and an associated unique barcode are assembled and delivered to cells using either in vivo or in vitro approaches. Cells or tissues are then prepared for sequential or multiplexed smFISH of the plasmid-derived mRNA transcripts that contain the unique enhancer barcodes, followed by spatial decoding of the self-transcribed enhancer mRNA transcripts and quantification of their activity. FIG. IB: Workflow for a single round of imaging for the barcoded transcripts. Adapter probes are first hybridized to a subset of the encoding probes that are bound to each barcoded transcript during sample preparation.Fluorescent readout probes are then hybridized to these adapter sequences, followed by imaging at three different wavelengths (750 nm, 647 nm, and 560 nm). Both the readout probes and adapters are subsequently stripped from the encoding probes with a formamide solution before starting the next imaging round. The encoding probes remain bound to the acrylamide gel due to an acrydite modification on the 5' ends of the probe sequences. The hybridization and imaging scheme is then repeated until all barcoded transcripts have been imaged in four of the 16 rounds of imaging. FIG. 1C: Representative diagram of the decoding scheme for barcoded enhancer transcripts using MERFISH 2.0. “On-bits” for each barcoded transcript are assigned according to which rounds of imaging they are detected in, resulting in a binary barcode for each transcript that contains four “on-bits” and 12 “off-bits.” The use of 3-color imaging effectively scales these binary barcodes to 48 bits, allowing for a robust error correction scheme.

[0010] FIGS. 2A-2D show precise quantification of enhancer activity at single-cell resolution using STARR-FISH. FIG. 2A: Representative smFISH data for three barcoded mRNA transcripts from HCT116 cells, and the resulting fitted spots assigned to segmentation masks. FIG. 2B: Relative enhancer activities in HCT116 cells. Error bars indicate the 95% confidence interval calculated using a simple linear regression. FIG. 2C: Pearson correlation between enhancer activities quantified via STARR-FISH and bulk RNA-seq. FIG. 2D: Pearson correlation between replicate experiments for enhancer activity as determined by STARR-FISH.

[0011] FIG. 3 shows multiplexed enhancer activity quantification via STARR-FISH requires less than one thousand cells. Enhancer activities calculated by randomly sampling 50-2500 cells from a single STARR-FISH experiment in HCT116 cells. Error bars indicate the 95% confidence interval calculated using a simple linear regression. The dashed line represents the coefficient of variation (C.V.) across ten random samplings.

[0012] FIGS. 4A-4B show STARR-FISH enables precise quantification of 300 CREs from a single experiment using MERFISH. FIG. 4A: Relative enhancer activities of 300 cCREs in HCT116 cells obtained with MERFISH. Error bars indicate the 95% confidence interval calculated using a simple linear regression. FIG. 4B: Pearson correlation between enhancer activities of 300 cCREs obtained from replicate experiments as determined by STARR-FISH.

[0013] FIG. 5 shows an exemplary’ overview of STARR-FISH (self-transcribing active regulatory region fluorescence in situ hybridization) method described herein. STARR-FISH is a reporter assay designed to screen candidate cis-regulatory elements, or enhancers, and quantify their capacity' to activate transcription from a minimally active promoter sequence using multiplexed error-robust FISH (MERFISH). STARR-FISH can quantify the activity of hundreds to thousands of enhancer sequences in parallel, at both single-cell and spatial resolution. (1) Plasmid libraries are first designed such that each plasmid contains a unique enhancer sequence and barcode sequence, which are assembled via a molecular cloning approach using synthetic DNA (See Figure 6). Importantly, a T7 promoter on the antisense strand downstream of the barcode sequence facilitates in situ transcription (1ST) during sample preparation. The plasmid libraries can then be directly transfected into cultured cells or packaged into AAV particles for deliver}' to animal tissues in vivo (2). There are two main sources of mRNA that are then detected in the assay. The first source is mRNA containing SYFP, enhancers, and their associated barcode sequences that are produced within living cells in a manner dependent on each enhancer’s transcriptional activity'. The second source is the antisense mRNA produced from the T7 1ST reaction during sample preparation post-fixation. (3) mRNA transcripts from both sources are then detected and quantified via MERFISH using fluorescently labeled DNA probes that primarily target the barcode sequences (both sense and antisense strands, see panel 1). In addition, cell ty pe marker gene transcripts can be quantified with MERFISH in parallel, providing detailed information about cell type identity within complex tissues. (4) Ultimately,normalized activity levels can be determined for each enhancer sequence using the T7-driven transcripts as an inferred copy number for each reporter construct, resulting in the identification of cell type-specific enhancer activity across complex tissues and unparalleled insight into gene regulator}' mechanisms associated with both development and disease.

[0014] FIGS. 6A-6D show an exemplary' method of STARR-FISH plasmid library design and assembly. FIG. 6A: cis-regulatory elements (cREs) are each assigned a unique 360 nucleotide barcode sequence prior to assembling the library. Each cRE-barcode DNA sequence is designed as two separate fragments initially, where the first fragment includes a short region homologous to the plasmid backbone (OH1), a cRE (typically <200 nucleotides), and a portion of the barcode sequence. The second fragment of the cRE-barcode sequence contains the rest of the barcode sequence, including ~40 nucleotides of homology with the 3’ end of the first fragment, and another short region homologous to the plasmid backbone (OH2). These cRE-barcode sequences are designed such that they can include hundreds to thousands of unique cRE-barcode combinations. FIG. 6B: The two fragments that comprise the full cRE-barcode sequence are synthesized as single stranded DNA oligos. The first fragment containing the cRE is synthesized 5’-3' as designed. However, the second fragment is synthesized 5'-3' as a reverse complement of the designed sequence. This ensures that the two single stranded DNA oligos are complementary to each other within the shared ~40 nucleotide overlap region of the barcode sequence. These single stranded DNA oligos, each between 300-350 nucleotides, can be synthesized as oligo pools from commercial vendors. The use of oligo pools in this process facilitates the assembly of hundreds to thousands of STARR-FISH plasmids in parallel. FIG. 6C: The single stranded fragments are assembled together using overlap extension PCR to create full length, double stranded cRE-barcode DNA cassettes. A second round of PCR using primers that target the 0H1 / 0H2 regions can then further amplify the double stranded cRE-barcode DNA cassettes. FIG. 6D: The pool of amplified cRE-barcode DNA cassettes are inserted into a digested plasmid backbone that contains the minimal CMV promoter, SYFP2 gene, and dow nstream poly adenylation sequence, using isothermal assembly (e.g., Gibson assembly).

[0015] FIGS. 7A-7H show that the method described herein (e.g., STARR-FISH) can precisely resolve enhancer activity at single-cell resolution in concordance with bulk sequencing assays. FIGS. 7A-7B: Boxplots showing average enhancer or T7 transcript counts per celldetected by STARR-FISH. HCT116 cells were transfected with a plasmid library containing 20 enhancer reporters and samples were prepared for imaging 48h post-transfection. Boxes represent the quartiles of the average count distributions per cell, while whiskers represent the full distribution for each reporter. FIGS. 7C-7D: Pearson correlation between biological replicates for either enhancer or T7 average transcripts per cell in transfected HCT116 cells. Dots represent log transformed counts for each of the 20 reporter transcripts. FIG. 7E: Pearson correlation between enhancer transcripts per cell detected by STARR-FISH and bulk RNA-seq counts for the same 20 reporter transcripts (cpm) in HCT116 cells. Dots represent log transformed counts for each of the 20 reporter transcripts. FIG. 7F: Pearson correlation between T7 transcripts per cell detected by STARR-FISH and bulk DNA-seq counts for the same 20 reporter transcripts (cpm) in HCT116 cells. Transfected plasmid DNA from the cells was extracted simultaneously with the RNA and PCR amplified prior to sequencing. Dots represent log transformed counts for each of the 20 reporter transcripts. FIG. 7G: STARR-FISH activity scores for the 20 reporter transcripts. Activity scores were calculated using a linear regression model, where enhancer transcripts per cell are normalized to the T7 transcripts from the same cells as a proxy for reporter copy number. Error bars represent the standard error of the mean. FIG. 7H: Pearson correlation between enhancer activities calculated from STARR-FISH and traditional (bulk sequencing-based) STARR-seq (Chen et al., 2022). Dots represent average activity measurements for each enhancer from the respective assays.

[0016] FIGS. 8A-8D show STARR-FISH enhancer activity can be normalized using either single-cell copy number data or total library complexity. FIG. 8A: STARR-FISH enhancer activity7quantification using single-cell normalization. Left: STARR-FISH activity scores for the 20 reporter transcripts. Activity scores were calculated using a linear regression model, where enhancer transcripts per cell are normalized to the T7 transcripts from the same cells as a proxy for reporter copy number. Error bars represent the standard error of the mean. Right: Pearson correlation between enhancer activities calculated from STARR-FISH and traditional (bulk sequencing-based) STARR-seq (Chen et al., 2022). Dots represent average activitymeasurements for each enhancer from the respective assays. FIG. 8B: STARR-FISH enhancer activity quantification using bulk library normalization. Left: STARR-FISH activity scores for the 20 reporter transcripts. Activity scores were calculated using a linear regression model,where enhancer transcripts per cell are normalized to total counts for each reporter as determined by long-read sequencing from the plasmid library used for transfection. Error bars represent the standard error of the mean. Right: Pearson correlation between enhancer activities calculated from STARR-FISH and traditional (bulk sequencing-based) STARR-seq (Chen et al., 2022). Dots represent average activity measurements for each enhancer from the respective assays. FIG. 8C: Downsampling for enhancer activity quantification using single-cell normalization. STARR-FISH activity scores for the 20 reporter transcripts (normalized to single-cell T7 transcript counts) were calculated using decreasing numbers of cells subsampled from the original dataset. Error bars represent the standard error of the mean obtained across 100 subsamplings. Dashed line represents the coefficient of variation (C.V.) among sampled means. FIG. 8D: Downsampling for enhancer activity quantification using bulk library normalization. STARR-FISH activity scores for the 20 reporter transcripts (normalized to total plasmid library counts for each reporter) were calculated using decreasing numbers of cells subsampled from the original dataset. Error bars represent the standard error of the mean obtained across 100 subsamplings. Dashed line represents the coefficient of variation (C.V.) among sampled means.

[0017] FIGS. 9A-9D show STARR-FISH can scale to hundreds of candidate cis-regulatory elements via MERFISH. FIG. 9A: STARR-FISH was performed in HCT116 cells using a plasmid library of 285 candidate cis-regulatory elements, or cCREs (enhancers). Transfected cells were fixed, hybridized with encoding probes targeting each of the enhancer-driven transcripts, and subjected to imaging via MERFISH. Representative images of barcoded enhancer transcripts captured during MERFISH imaging are shown. Transcripts were imaged in a predetermined order, where appropriate adapters are hybridized to the sample in four out of sixteen rounds of imaging. Circled spots represent barcoded enhancer mRNA transcripts detected in four rounds of imaging as expected. Each detected spot corresponds to a “I ” in a binary code, whereas each absence of the same spot corresponds to a “0” within the same binary code. The binary code containing four “on bits” can be decoded to reveal the identity of each transcript. For example, here the circled spots correspond to the enhancer reporter-driven transcript CRE094. FIG. 9B: Quantification of STARR-FISH activity for 285 different cCREs captured within a single experiment. Each dot represents a different CRE transcript. Gray outline represents standard error derived from activity quantification in each single cell. FIG. 9C:Pearson correlation between two biological replicates of STARR-FISH for the 285 cCREs. FIG. 9D: The same plasmid library of 285 cCREs was transfected into a human induced pluripotent stem cell line, WTC11. STARR-FISH was carried out as in the HCT116 cells to quantify enhancer activity. The 295 cCREs were separated into 3 clusters based on their activity in each cell line, corresponding to enhancers whose activity was enriched in 1) HCT116 cells, 2) WTCI I cells, or 3) neither cell line. These results demonstrate the capacity of STARR-FISH to resolve cell type-specific enhancer activity. P values were calculated via Student’s t-test.

[0018] FIGS. 10A-10C show STARR-FISH can be performed in vivo to quantify' cell typespecific activity' for hundreds of enhancers in the whole mouse brain. FIG. 10A: Overview of in vivo STARR-FISH experiment in the mouse brain. 400 cCREs were selected based on predicted activity across a wide array of cell types (subclasses) and neurotransmitter types. Ten negative control sequences (designed to contain no known transcription factor motifs) were used for normalization purposes. A pooled library' of plasmids for all 400 cCRE STARR-FISH reporter constructs was packaged into AAV-PHP.eB particles and delivered retro-orbi tally into 7-week old C57BL / 6 mice. Mouse brains were collected and sectioned in preparation for STARR-FISH, consisting of MERFISH imaging for all 400 barcoded cCRE transcripts as well as 500 marker gene transcripts for cell type identification. FIG. 10B: Dot plot showing enhancer activities for a subset of the 400 cCREs that showed significant and selective activity' within identified cell types. FIG. 10C: Example spatial data for CRE004 activity as determined by STARR-FISH (top) and the corresponding cell types where CRE004 activity was significantly enriched (bottom). Each point represents a single cell within the mouse brain.DETAILED DESCRIPTIONDEFINITIONS

[0019] While various embodiments and aspects of the present invention are show n and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0020] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

[0021] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0022] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed„ J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0023] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof; or nucleosides (e.g., deoxyribonucleosides or ribonucleosides). In embodiments, “nucleic acid” does not include nucleosides. The terms “polynucleotide,” “oligonucleotide,” “oligo" or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term “nucleoside” refers, in the usual and customary sense, to a glycosylamine including a nucleobase and a five-carbon sugar (ribose or deoxyribose). Non limiting examples, of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acid, e.g. polynucleotides contemplated herein include any types of RNA. e.g. mRNA, siRNA. miRNA, and guide RNA and any types of DNA, genomic DNA,plasmid DNA, and minicircle DNA, and any fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

[0024] Nucleic acids, including e.g., nucleic acids with a phosphothioate backbone, can include one or more reactive moieties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, e.g., a nucleic acid or polypeptide through covalent, non-covalent or other interactions. By way of example, the nucleic acid can include an amino acid reactive moiety that reacts with an amino acid on a protein or polypeptide through a covalent, non-covalent or other interaction.

[0025] The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, OxfordUniversity Press) as well as modifications to the nucleotide bases such as in 5-methyl cytidine or pseudouridine.; and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-iomc backbones, modified sugars, and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA) as known in the art), including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphatebackbone may be done for a variety of reasons, e.g.. to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In embodiments, the intemucleotide linkages in DNA are phosphodiester, phosphodi ester derivatives, or a combination of both.

[0026] Nucleic acids can include nonspecific sequences. As used herein, the term "nonspecific sequence" refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to or are only partially complementary to any other nucleic acid sequence. By way of example, a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism.

[0027] A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term "‘polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homolog}' searching. Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and / or modified nucleotides.

[0028] The term “complement,” as used herein, refers to a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides capable of base pairing with a complementary nucleotide or sequence of nucleotides. As described herein and commonly known in the art the complementary (matching) nucleotide of adenosine is thymidine and the complementary' (matching) nucleotide of guanosine is cytosine. Thus, a complement may include a sequence of nucleotides that base pair with corresponding complementary nucleotides of a second nucleic acid sequence. The nucleotides of a complement may' partially or completely' match the nucleotides of the second nucleic acid sequence. Where the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. Where the nucleotides of the complement partially match thenucleotides of the second nucleic acid sequence only some of the nucleotides of the complement form base pairs with nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding and a non-coding sequences, wherein the non-coding sequence contains complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence. A further example of complementary sequences are sense and antisense sequences, wherein the sense sequence contains complementary nucleotides to the antisense sequence and thus forms the complement of the antisense sequence.

[0029] As described herein the complementarity of sequences may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing. Thus, two sequences that are complementary’ to each other, may have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region).

[0030] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxy glutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring ammo acid. The terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

[0031] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB BiochemicalNomenclature Commission. Nucleotides, likewise, may be referred to by their commonly- accepted single-letter codes.

[0032] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may In embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0033] An amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be considered when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0034] The terms "numbered with reference to" or "corresponding to," when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. An amino acid residue in a protein "corresponds" to a given residue when it occupies the same essential structural position within the protein as the given residue. One skilled in the art will immediately recognize the identity and location of residues corresponding to a specific position in a protein (e g., a transcription factor) in other proteins with different numbering systems. For example, byperforming a simple sequence alignment with a protein (e.g., transcription factor) the identity’ and location of residues corresponding to specific positions of the protein are identified in other protein sequences aligning to the protein. For example, a selected residue in a selected protein corresponds to glutamic acid at position 138 when the selected residue occupies the same essential spatial or other structural relationship as a glutamic acid at position 138. In some embodiments, where a selected protein is aligned for maximum homology with a protein, the position in the aligned selected protein aligning with glutamic acid 138 is the to correspond to glutamic acid 138. Instead of a primary' sequence alignment, a three dimensional structural alignment can also be used, e.g., where the structure of the selected protein is aligned for maximum correspondence with the glutamic acid at position 138, and the overall structures compared. In this case, an amino acid that occupies the same essential position as glutamic acid 138 in the structural model is said to correspond to the glutamic acid 138 residue.

[0035] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences w ill encode any' given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every' position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations." which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every' possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG. which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0036] As to amino acid sequences, one of skill w ill recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an aminoacid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure.

[0037] The following eight groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M)(see, e.g., Creighton, Proteins (1984)).

[0038] The terms "identical" or percent "identity ," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http: / / www.ncbi.nlm.nih.gov / BLAST / or the like). Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, aswell as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0039] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0040] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of. e.g., a full length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group. 575 Science Dr.. Madison. Wl). or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0041] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol.215:403-410. respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantify X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3. and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0042] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see. e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0043] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0044] The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory' Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0045] A "ligand" refers to an agent, e.g., a polypeptide or other molecule, capable of binding to a receptor or antibody, antibody variant, antibody region or fragment thereof.

[0046] Techniques for conjugating therapeutic agents to antibodies are well known (see, e.g., Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., ‘"Antibodies For Drug Delivery”in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker. Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review" in Monoclonal Antibodies ‘84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62: 119- 58 (1982)). As used herein, the term “antibody-drug conjugate” or “ADC” refers to a therapeutic agent conjugated or otherwise covalently bound to to an antibody.

[0047] For specific proteins described herein, the named protein includes any of the protein’s naturally occurring forms, variants or homologs that maintain the protein transcription factor activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity- compared to the native protein). In some embodiments, variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form. In other embodiments, the protein is the protein as identified by its NCBI sequence reference. In other embodiments, the protein is the protein as identified by its NCBI sequence reference, homolog or functional fragment thereof.

[0048] The term "gene" means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary- during the transcription and the translation of a gene. Further, a "protein gene product" is a protein expressed from a particular gene.

[0049] The term “spacer domain” is used herein according to its plain ordinary meaning and refers to a nucleic acid sequence that separates two nucleic acid sequences.

[0050] The terms "plasmid", "vector" or "expression vector" refer to a nucleic acid molecule that encodes for genes and / or regulatory elements necessary for the expression of genes. Expression of a gene from a plasmid can occur in cis or in trans. If a gene is expressed in cis. the gene and the regulatory elements are encoded by the same plasmid. Expression in trans refers to the instance where the gene and the regulatory- elements are encoded by separate plasmids.

[0051] The terms "transfection", "transduction", "transfecting" or "transducing" can be used interchangeably and are defined as a process of introducing a nucleic acid molecule or a protein to a cell. Nucleic acids are introduced to a cell using non-viral or viral-based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. Non-viral methods of transfection include any appropriate transfection method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non-viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heat shock, magnetifection and electroporation. In some embodiments, the nucleic acid molecules are introduced into a cell using electroporation following standard procedures well known in the art. For viral-based methods of transfection any useful viral vector may be used in the methods described herein. Examples for viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors. In some embodiments, the nucleic acid molecules are introduced into a cell using a retroviral vector following standard procedures well know n in the art. The terms "transfection" or "transduction" also refer to introducing proteins into a cell from the external environment. Typically, transduction or transfection of a protein relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g., Ford et al. (2001) Gene Therapy 8: 1-4 and Prochiantz (2007) Nat. Methods 4: 119-20.

[0052] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. Any appropriate method known in the art for conjugating an antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

[0053] When the label or detectable moiety is a radioactive metal or paramagnetic ion, the agent may be reacted wi th another long-tailed reagent having a long tail with one or more chelating groups attached to the long tail for binding to these ions. The long tail may be apolymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which the metals or ions may be added for binding. Examples of chelating groups that may be used according to the disclosure include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTP A), DOT A, NOTA, NET A. TETA, porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, poly oximes, and like groups. The chelate is normally linked to the PSMA antibody or functional antibody fragment by a group, which enables the formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking. The same chelates, when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI, when used along with the antibodies and carriers described herein. Macrocyclic chelates such as NOTA, DOT A, and TETA are of use with a variety of metals and radiometals including, but not limited to, radionuclides of gallium, yttrium and copper, respectively. Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as223Ra for RAIT may be used. In certain embodiments, chelating moieties may be used to attach a PET imaging agent, such as an A1-18F complex, to a targeting molecule for use in PET analysis.

[0054] "Contacting" is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e g. antibodies and antigens) to become sufficiently proximal to react, interact, or physically touch. It should be appreciated, however, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

[0055] The term "contacting" may include allowing two species to react, interact, or physically touch, wherein the two species may be, for example, a pharmaceutical composition as provided herein and a cell. In embodiments contacting includes, for example, allowing a pharmaceutical composition as described herein to interact with a cell.

[0056] A "cell" as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability' to combine with asecond gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokar otic cells include but are not limited to bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells.

[0057] The term "recombinant" when used with reference, e.g., to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. Transgenic cells and plants are those that express a heterologous gene or coding sequence, typically as a result of recombinant methods.

[0058] The term "isolated", when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0059] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g.. a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0060] The term "exogenous" refers to a molecule or substance (e.g., a compound, nucleic acid or protein) that originates from outside a given cell or organism. For example, an "exogenouspromoter" as referred to herein is a promoter that does not originate from the cell or organism it is expressed by. Conversely, the term "endogenous" or "endogenous promoter" refers to a molecule or substance that is native to, or originates within, a given cell or organism.

[0061] As defined herein, the term "inhibition", "inhibit", "inhibiting" and the like in reference to cell proliferation (e.g., cancer cell proliferation) means negatively affecting (e.g., decreasing proliferation) or killing the cell. In embodiments, inhibition refers to reduction of a disease or symptoms of disease (e.g., cancer, cancer cell proliferation). Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enz matic activity' or the amount of a protein (e.g.. a cancer-associated protein). Similarly, an "inhibitor" is a compound or protein that inhibits a receptor or another protein, e.g.., by binding, partially or totally blocking, decreasing, preventing, delaying, inactivating, desensitizing, or down -regulating activity (e.g., a receptor activity or a protein activity).

[0062] The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0063] ‘‘Biological sample" or “sample” refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells) stool, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. A biological sample is typically obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.

[0064] A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g. cancer) and compared to a known normal (non-diseased) individual (e.g. a standard control subject). A standard control can also represent an average measurement or value gathered from a population of similar individuals (e.g. standard control subjects) that do not have a given disease (i.e. standard control population), e.g., healthy individuals with a similar medical background, same age, weight, etc. A standard control value can also be obtained from the same individual, e.g. from an earlier-obtained sample from the patient prior to disease onset. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., halflife) or therapeutic measures (e.g., comparison of side effects). Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g. RNA levels, protein levels, specific cell types, specific bodily fluids, specific tissues, etc).

[0065] The term “cis-regulatory element” or “CRE” is used herein according to its plain ordinary' meaning and refers to a region of non-coding DNA which regulates transcription of a gene. In embodiments, cis-regulatory element and cis-regulator module (CRM) are used interchangeably. In embodiments, the CRE regulates the transcription of a neighboring gene. In embodiments, the CRE binds to a transcription factor. In embodiments, the CRE is between about 100 to about 1000 nucleotides in length.

[0066] The term “transcriptional activity ” is used herein according to its plain and ordinary meaning and refers to the process of transcribing DNA (e.g., a gene) into RNA (e.g., messenger RNA). In embodiments, the transcriptional activity of a biomolecule includes how the biomolecule modulates (e g., increases and / or decreases) transcription of a DNA sequence. In embodiments, the transcriptional activity of an enhancer is the level of transcription of a nucleic acid (e.g., DNA, a gene) in the presence of the enhancer relative to the level of transcription of the nucleic acid in the absence of the enhancer. Techniques for assaying transcriptional activity of a cis regulatory element (e.g., an enhancer) are well known in the art. For example. Self-Transcribing Active Regulatory Region sequencing (STARRseq) is a well-known reporter assay to identify transcriptional enhancers. See, e.g., Arnold et al., Science, 339, 6123 (2013), which is incorporated herein in its entirety and for all purposes.

[0067] The term “enhancer” is used herein according to its plain and ordinary meaning and refers to a region of DNA that may be bound by proteins (e.g., transcription factors) to increase the likelihood that transcription of a gene will occur. In embodiments, enhancers may be about 50 to about 1500 base pairs in length. In embodiments, enhancers may be located downstream or upstream of the transcription initiation site that it regulates and may be several hundreds of base pairs, several thousands of base pairs, or several millions of base pairs away from the transcription initiation site. In embodiments, the enhancer sequence includes the nucleotide sequence of any one of SEQ ID NOs: 1-40. In embodiments, the enhancer sequence has the nucleotide sequence of any one of SEQ ID NOs: 1-40. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 1. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:2. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:3. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:4. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:5. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 6. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 7. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 8. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 9. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NOTO. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 11. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 12. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 13. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 14. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 15. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 16. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 17. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 18. In embodiments, the enhancer sequenceincludes the nucleotide sequence of SEQ ID NO: 19. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:20. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:21. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:22. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:23. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:24. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:25. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:26. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:27. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:28. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:29. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 30. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:31. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 32. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:33. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 34. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:35. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 36. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:37. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:38. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO: 39. In embodiments, the enhancer sequence includes the nucleotide sequence of SEQ ID NO:40.

[0068] The term ‘‘barcode sequence'’ is used herein according to its plain and ordinary meaning and refers to a nucleic acid sequence that is used to identify a nucleic acid (e.g., a DNA sequence or an RNA transcript). In embodiments, the barcode sequence is operably linked to an enhancer sequence. In embodiments, the barcode sequence identifies the enhancer sequence. In embodiments, the barcode sequence identifies the RNA transcript. In embodiments, the barcode sequence is 360 nucleotides in length. In embodiments, the barcode sequence includes 9 barcode subsequences. In embodiments, each barcode subsequence unit is 40 nucleotides in length. In embodiments, the barcode subsequence is complementary to a barcode-binding sequence withinan encoding probe. In embodiments, the barcode subsequence is capable of hybridizing with the complementary barcode-binding sequence within the encoding probe. In embodiments, each of the 9 barcode subsequences are capable of hybridizing to a different barcode-binding sequence.

[0069] The term “operably linked” is used herein according to its plain and ordinary meaning and refers to a physical or functional linkage between two or more elements, e.g., polynucleotide sequences, which permits them to operate in their intended fashion. In embodiments, the operably linkage between a polynucleotide of interest (e.g., barcode sequence) and a regulatory sequence (e.g., an enhancer) is functional link that allows for expression of the polynucleotide of interest. In embodiments, the term "operably linked" refers to the positioning of a regulatory7region (e.g. an enhancer sequence) and a coding sequence (e.g., a barcode sequence) to be transcribed so that the regulatory region is effective for regulating transcription or translation of the coding sequence of interest. In embodiments, the term "operably linked” denotes a configuration in which a regulatory' sequence (e.g., an enhancer sequence) is placed at an appropriate position relative to a sequence that encodes an RNA transcript (e.g., barcode sequence) such that the control sequence directs or regulates the expression of the mRNA encoding the RNA transcript. In embodiments, an enhancer sequence is in operable linkage with a barcode sequence if it can mediate transcription of the barcode sequence. In embodiments, operably linked elements may be contiguous or non-contiguous.

[0070] The term “barcoded RNA transcript” is used herein according to its plain and ordinary7meaning and refers to a transcribed RNA sequence that includes a barcode sequence.

[0071] The term “encoding probe” is used herein according to its plain and ordinary meaning and refers to a nucleic acid sequence including a barcode-binding sequence and a first adapterbinding sequence. In embodiments, the encoding probe is capable of hybridizing to a barcoded RNA transcript. In embodiments, the encoding probe is capable of hybridizing to a complementary barcode subsequence within a barcoded RNA transcript. In embodiments, the encoding probe is capable of hybridizing to a complementary barcode subsequence within a barcode sequence. In embodiments, the encoding probe is capable of hybridizing to an adapter probe. In embodiments, the encoding probe further includes a second adapter probe-binding sequence and / or a third adapter probe-binding sequence. In embodiments, the encoding probe is an acrydite-modified encoding probe. In embodiments, the encoding probe includes thenucleotide sequence of any one of SEQ ID NOs:41-220. In embodiments, the encoding probe has the nucleotide sequence of any one of SEQ ID NOs:41-220.

[0072] The term “acrydite-modified encoding probe” is used herein according to its plain and ordinary meaning and refers to an encoding probe with an aery di te modification on the 5' end. In embodiments, the acrydite modification includes addition of an acry lic phosphoramidite. In embodiments, an acrydite-modified encoding probe is capable of polymerization into an acry lamide co-poly er (e.g., an acrylamide gel). In embodiments, the polymerization attaches the acry dite-modified encoding probe to the acrylamide gel.

[0073] The term “encoding probe RNA transcript hybridized complex” is used herein according to its plain and ordinary meaning and refers to a complex formed by the hybridization of a plurality of encoding probes to a barcoded RNA transcript.

[0074] The term “barcode-binding sequence” is used herein according to its plain and ordinary meaning and refers to a nucleic acid sequence within an encoding probe that is capable of hybridizing to a barcoded RNA transcript. In embodiments, the barcode-binding sequence is capable of hybridizing to a complementary barcode subsequence within a barcoded RNA transcript. In embodiments, the barcode-binding sequence is capable of hybridizing to a barcode sequence. In embodiments, the barcode-binding sequence is capable of hybridizing to a complementary7barcode subsequence within a barcode sequence.

[0075] The term “adapter probe-binding sequence” is used herein according to its plain and ordinary7meaning and refers to a nucleic acid sequence within an encoding probe that is capable of hybridizing to an adapter probe. In embodiments, the adapter probe-binding sequence is capable of hybridizing to a complementary subsequence within an adapter probe. In embodiments, the adapter probe-binding sequence is capable of hybridizing to a complementary encoding probe-binding sequence within the adapter probe.

[0076] The term “adapter probe” is used herein according to its plain and ordinary7meaning and refers to a nucleic acid sequence including a first encoding probe-binding sequence and a first fluorescent readout probe-binding sequence. In embodiments, the adapter probe is capable of hybridizing to an encoding probe RNA transcript complex. In embodiments, the adapter probe is capable of hybridizing to an encoding probe. In embodiments, the adapter probe is capable ofhybridizing to a complementary subsequence within the encoding probe. In embodiments, the adapter probe is capable of hybridizing to a complementary adapter probe-binding sequence within the encoding probe. In embodiments, the encoding probe further includes a second encoding probe-binding sequence and / or a third encoding probe-binding sequence. In embodiments, the adapter probe is capable of hybridizing to a fluorescence readout probe. In embodiments, the adapter probe further includes a second fluorescent readout probe-binding sequence. In embodiments, the adapter probe includes the nucleotide sequence of any one of SEQ ID N0s:381-400. In embodiments, the adapter probe has the nucleotide sequence of any one of SEQ ID N0s:381-400. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:381. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:382. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:383. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:384. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:385. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:386. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:387. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO: 388. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:389. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:390. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:390. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:392. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:393. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:394. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:395. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:396. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:397. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:398. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:399. In embodiments, the adapter probe includes the nucleotide sequence of SEQ ID NO:400.

[0077] The term "‘encoding probe-binding sequence" is used herein according to its plain and ordinary meaning and refers to a nucleic acid sequence within an adapter probe that is capable of hybridizing to an encoding probe. In embodiments, the encoding probe-binding sequence is capable of hybridizing to a complementary subsequence within an encoding probe. In embodiments, the encoding probe-binding sequence is capable of hybridizing to a complementary adapter probe-binding sequence within the encoding probe.

[0078] The term “adapter probe RNA transcript hybridized complex” is used herein according to its plain and ordinary meaning and refers to a complex formed by the hybridization of a plurality' of adapter probes to an encoding probe RNA transcript hybridized complex.

[0079] The term “fluorescent readout probe-binding sequence” is used herein according to its plain and ordinary meaning and refers to a nucleic acid sequence within an adapter probe that is capable of hybridizing to a fluorescent readout probe. In embodiments, the fluorescent readout probe-binding sequence is capable of hybridizing to a complementary subsequence within the adapter probe.

[0080] The term “fluorescent readout probe RNA transcript hybridized complex” is used herein according to its plain and ordinary’ meaning and refers to a complex formed by the hybridization of a plurality of fluorescent readout probes to an adapter probe RNA transcript hybridized complex.

[0081] The term “fluorescent readout probe” is used herein according to its plain and ordinary meaning and refers to a nucleic acid sequence including a complementary fluorescent readout probe-binding sequence and a chemical fluorophore. In embodiments, the fluorescent readout probe is capable of hybridizing to an adapter probe RNA transcript complex. In embodiments, the fluorescent readout probe is capable of hybridizing to an adapter probe. In embodiments, the fluorescent readout probe is capable of hybridizing to a fluorescent readout probe-binding sequence within the adapter probe. In embodiments, the complementary fluorescent readout probe-binding sequence is capable of hybridizing to a fluorescent readout probe binding sequence within the adapter probe. In embodiments, the complementary fluorescent readout probe-binding sequence includes the nucleotide sequence of any one of SEQ ID NOs:421-423. In embodiments, the complementary fluorescent readout probe-binding sequence has thenucleotide sequence of any one of SEQ ID NOs:421-423. In embodiments, the complementary fluorescent readout probe-binding sequence includes the nucleotide sequence of SEQ ID NO:421. In embodiments, the complementary fluorescent readout probe-binding sequence includes the nucleotide sequence of SEQ ID NO:422. In embodiments, the complementary fluorescent readout probe-binding sequence includes the nucleotide sequence of SEQ ID NO:423. In embodiments, the complementary fluorescent readout probe-binding sequence includes the nucleotide sequence of SEQ ID NO:421 and the chemical fluorophore is an Alexa 750 fluorophore. In embodiments, the complementary fluorescent readout probe-binding sequence includes the nucleotide sequence of SEQ ID NO: 422 and the chemical fluorophore is Cy5 fluorophore. In embodiments, the complementary fluorescent readout probe-binding sequence includes the nucleotide sequence of SEQ ID NO:423 and the chemical fluorophore is Cy3 fluorophore.

[0082] The term “chemical fluorophore” is used herein according to its plain and ordinary meaning and refers to a light-sensitive chemical compound that can re-emit light upon light excitation. In embodiments, the chemical fluorophore is an Alexa Fluor 750 fluorophore. a Cy5 fluorophore, a Cy3 fluorophore, a 6-FAM fluorophore, a 6-FAM fluorophore, a Fluorescein dT fluorophore, a TAMRA fluorophore, a JOE fluorophore, a MAX fluorophore, a TET fluorophore, a TET fluorophore, a Cy5.5 fluorophore, a ROX fluorophore, a TYE 563 fluorophore, a Yakima Yellow fluorophore, a HEX fluorophore, a TEX 615 fluorophore, a TYE 665 fluorophore, a TYE 705 fluorophore, a SUN fluorophore. an ATTO 425 fluorophore, an ATTO 488 fluorophore, an ATTO 532 fluorophore, an ATTO 550 fluorophore, an ATTO 565 fluorophore, an ATTO RholOl fluorophore, an ATTO 590 fluorophore, an ATTO 633 fluorophore, an ATTO 647N fluorophore, an ATTO 700 fluorophore, an Alex Fluor 405 fluorophore, an Alex Fluor 488 fluorophore. an Alex Fluor 532 fluorophore, an Alex Fluor 546 fluorophore, an Alex Fluor 594 fluorophore, an Alex Fluor 647 fluorophore, an Alex Fluor 660 fluorophore, a 5' IRDye 700 fluorophore, a 5' IRDye 800 fluorophore, a 5' IRDye 800CW fluorophore, a Rhodamine Green-X fluorophore, a Rhodamine Red fluorophore, a 5-TAMRA fluorophore, a Texas Red-X fluorophore, a Lightcycler 640 fluorophore, or a Dy 750 fluorophore.

[0083] The term “T7 polymerase" is used herein according to its plain and ordinary meaning and refers to an RNA polymerase from the T7 bacteriophage that catalyzes the transcription of a DNA sequence into an RNA transcript. In embodiments, the T7 polymerase transcribes the DNA sequence to produce an antisense barcode transcript. In embodiments, the T7 polymerase- mediated transcription is promoter-specific. In embodiments, the T7 polymerase-mediated transcription only occurs downstream of a T7 polymerase promoter.

[0084] The term “T7 polymerase promoter” is used herein according to its plain and ordinary meaning and refers to a DNA sequence that is the transcription initiation site for a T7 RNA polymerase.

[0085] The term ‘‘promoter” is used herein according to its plain and ordinary' meaning and refers to a DNA sequence that is the transcription initiation site for a polymerase. In embodiments, the promoter is a minimal promoter. In embodiments, the minimal promoter is a truncated promoter. In embodiments, the minimal promoter contains the minimal elements for initiation of transcription. In embodiments, the minimal promoter is a cytomegalovirus (CMV) minimal promoter.

[0086] The term “antisense barcode transcript” is used herein according to its plain and ordinary meaning and refers to a nucleic acid sequence produced by T7 polymerase-mediated transcription of the barcode sequence. In embodiments, the antisense barcode transcript is an antisense strand complementary' to the barcode sequence.

[0087] The term “antisense encoding probe” is used herein according to its plain and ordinary' meaning and refers to nucleic acid sequence including an antisense barcode-binding sequence and an antisense adapter-binding sequence. In embodiments, the antisense encoding probe is capable of hybridizing to an antisense barcode transcript. In embodiments, the antisense encoding probe is capable of hybridizing to a complementary' antisense barcode subsequence within an antisense barcode transcript. In embodiments, the antisense encoding probe is capable of hybridizing to a complementary antisense barcode subsequence within an antisense barcode sequence. In embodiments, the e antisense encoding probe is capable of hybridizing to an antisense adapter probe. In embodiments, the antisense encoding probe further includes a second antisense adapter probe-binding sequence and / or a third antisense adapter probe-bindingsequence. In embodiments, the antisense encoding probe includes an acrydite modification on the 5' end. In embodiments, the antisense encoding probe includes the nucleotide sequence of any one of SEQ ID NOs:221-380. In embodiments, the encoding probe has the nucleotide sequence of any one of SEQ ID NOs:221-380.

[0088] The term ‘‘encoding probe antisense barcode transcript hybridized complex'’ is used herein according to its plain and ordinary meaning and refers to a complex formed by the hybridization of a plurality of antisense encoding probes to an antisense barcode transcript.

[0089] The term “antisense barcode-binding sequence” is used herein according to its plain and ordinary meaning and refers to a nucleic acid sequence within an antisense encoding probe that is capable of hybridizing to an antisense barcode transcript. In embodiments, the antisense barcode-binding sequence is capable of hybridizing to a complementary antisense barcode subsequence within an antisense barcode transcript. In embodiments, the antisense barcodebinding sequence is capable of hybridizing to an antisense barcode sequence. In embodiments, the antisense barcode-binding sequence is capable of hybridizing to a complementary' antisense barcode subsequence within an antisense barcode sequence.

[0090] The term “antisense adapter probe-binding sequence” is used herein according to its plain and ordinary meaning and refers to a nucleic acid sequence within an antisense encoding probe that is capable of hybridizing to an antisense adapter probe. In embodiments, the antisense adapter probe-binding sequence is capable of hybridizing to a complementary subsequence within an antisense adapter probe. In embodiments, the antisense adapter probe-binding sequence is capable of hybridizing to a complementary antisense encoding probe-binding sequence within the antisense adapter probe.

[0091] The term “antisense adapter probe” is used herein according to its plain and ordinary meaning and refers to a nucleic acid sequence including a first antisense encoding probe-binding sequence and a first antisense fluorescent readout probe-binding sequence. In embodiments, the antisense adapter probe is capable of hybridizing to an antisense encoding probe RNA transcript complex. In embodiments, the antisense adapter probe is capable of hybridizing to an antisense encoding probe. In embodiments, the antisense adapter probe is capable of hybridizing to a complementary subsequence within the antisense encoding probe. In embodiments, the antisenseadapter probe is capable of hybridizing to a complementary antisense adapter probe-binding sequence within the antisense encoding probe. In embodiments, the antisense encoding probe further includes a second antisense encoding probe-binding sequence and / or a third antisense encoding probe-binding sequence. In embodiments, the antisense adapter probe is capable of hybridizing to a fluorescence readout probe. In embodiments, the antisense adapter probe further includes a second antisense fluorescent readout probe-binding sequence. In embodiments, the antisense adapter probe includes the nucleotide sequence of any one of SEQ ID NOs:401-420. In embodiments, the antisense adapter probe has the nucleotide sequence of any one of SEQ ID N0s:401-420. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:401. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:402. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:403. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:404. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:405. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:406. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:407. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO: 408. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:409. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:410. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:411. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:412. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NON 13. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:414. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:415. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:416. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:417. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:418. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:419. In embodiments, the antisense adapter probe includes the nucleotide sequence of SEQ ID NO:420.

[0092] The term "‘antisense encoding probe-binding sequence" is used herein according to its plain and ordinary meaning and refers to a nucleic acid sequence within an antisense adapter probe that is capable of hybridizing to an antisense encoding probe. In embodiments, the antisense encoding probe-binding sequence is capable of hybridizing to a complementary subsequence within an antisense encoding probe. In embodiments, the antisense encoding probebinding sequence is capable of hybridizing to a complementary antisense adapter probe-binding sequence within the antisense encoding probe.

[0093] The term “adapter probe antisense barcode transcript hybridized complex” is used herein according to its plain and ordinary meaning and refers to a complex formed by the hybridization of a plurality of antisense adapter probes to an encoding probe antisense barcode transcript hybridized complex.

[0094] The term “antisense fluorescent readout probe-binding sequence” is used herein according to its plain and ordinary meaning and refers to a nucleic acid sequence within an antisense adapter probe that is capable of hybridizing to a fluorescent readout probe. In embodiments, the antisense fluorescent readout probe-binding sequence is capable of hybridizing to a complementary subsequence within the adapter probe.

[0095] The term “fluorescent readout probe antisense barcode transcript hybridized complex” is used herein according to its plain and ordinary meaning and refers to a complex formed by the hybridization of a plurality' of fluorescent readout probes to an adapter probe antisense barcode transcript hybridized complex.

[0096] The term “hydrogel” is used herein according to its plain and ordinary meaning and refers to three-dimensional polymer network capable of absorbing water. In embodiments, the three-dimensional polymer network includes physically or chemically interconnected (e.g. crosslinked) hydrophilic polymers. In embodiments, the three-dimensional polymer network includes natural and / or synthetic hydrophilic polymeric chains. In embodiments, the hydrogel includes hydrophilic polymeric chains of various lengths and chemical compositions. In embodiments, the hydrophilic polymeric chains include monomers, oligomers, or blockpolymeric units interconnected (e.g., crosslinked) by chemical bonds (e.g., covalent, hydrogen, ionic, complex, and / or metallic bonds). In embodiments, the hydrogel further includes water. Inembodiments, the hydrogel includes at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (by weight) water. In embodiments, the hydrogel is an acrylamide gel. In embodiments, the hydrogel is a polyacrylamide gel.

[0097] The term ‘‘probe-stripping buffer” is used herein according to its plain and ordinary meaning and refers to a solution capable of separating (e.g., denaturing) hybridized nucleic acid sequences. In embodiments the probe-stripping buffer is capable of denaturing nucleic acid probes from hybridized complexes. In embodiments, the probe-stripping buffer is capable of denaturing an adapter probe hybridized to an encoding probe RNA transcript hybridized complex, thereby producing a free adapter probe. In embodiments, the probe-stripping buffer is capable of denaturing a fluorescent readout probe hybridized to an adapter probe RNA transcript hybridized complex, thereby producing a free fluorescent readout probe. In embodiments, the probe-stripping buffer is not capable of denaturing an encoding probe hybridized to a barcoded RNA transcript. In embodiments, the probe-stripping buffer is not capable of denaturing an acrydite-modified encoding probe polymerized into an acrylamide gel. In embodiments, the probe-stripping buffer includes formamide and / or saline-sodium citrate (SSC). In embodiments, the probe-stripping buffer includes about 80% formamide and about 0.8X SSC.

[0098] One of skill in the art will understand which standard controls are most appropriate in a given situation and be able to analyze data based on comparisons to standard control values. Standard controls are also valuable for determining the significance (e.g. statistical significance) of data. For example, if values for a given parameter are widely variant in standard controls, variation in test samples will not be considered as significant.

[0099] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.METHODS

[0100] Compositions and kits provided herein including embodiments thereof may be used, inter alia, to detect transcriptional activity of a candidate cis regulatory element (e.g., an enhancer). Provided herein is a spatially resolved, single cell-based, high-throughput reporter assay to detect transcriptional activity of a candidate cis regulatory element. The methods provided herein including embodiments thereof combine an ultra-high throughput assay for testing enhancer activities with multiplexed error-robust fluorescence in situ hybridization for single cell spatial transcriptomics. The methods provided herein including embodiments thereof are useful, inter alia, for investigating gene regulation in health and disease. Thus, in an aspect is provided a method of detecting transcriptional activity of an enhancer sequence in a cell, the method including: (a) delivering a plasmid to a cell, wherein the plasmid includes a DNA sequence including a protein encoding sequence, an enhancer sequence and a barcode sequence associated with the enhancer sequence and allowing the cell to transcribe the plasmid thereby producing a barcoded RNA transcript including the protein encoding sequence, the enhancer sequence, and the barcode sequence, wherein the barcode sequence is operably linked to the enhancer sequence; (b) fixing the cell; (c) contacting the barcoded RNA transcript with a plurality of different encoding probes and allowing each of the plurality of different encoding probes to hybridize to a different subsequence within the barcoded RNA transcript thereby forming a plurality of encoding probe RNA transcript hybridized complexes; (d) contacting the plurality of encoding probe RNA transcript hybridized complexes with a first plurality of different adapter probes and allowing each of the different adapter probe in the first plurality of different adapter probes to hybridize to a different encoding probe bound to the barcoded RNA transcript thereby forming a first plurality of adapter probe RNA transcript hybridized complexes; (e) contacting the first plurality of adapter probe RNA transcript hybridized complexes with a plurality of different fluorescent readout probes and allowing each of the different fluorescent readout probes to hybridize to a different adapter probe bound to the barcoded RNA transcript thereby forming a first plurality of fluorescent readout probe RNA transcript hybridized complexes; and (f) detecting the first plurality of different fluorescent readout probes hybridized to the barcoded RNA transcript, thereby detecting the barcoded RNA transcript and the enhancer transcriptional activity.

[0101] In embodiments, the method provided herein enables simultaneous analysis of the transcriptional activity of a large number of candidate cis regulatory elements (e.g., enhancers) at single-cell resolution. In embodiments, the method provided herein includes reporter constructs (e.g., plasmids and / or DNA sequences) that link candidate cis regulatory elements (e.g., enhancers) with molecular barcode sequences for fluorescent in situ hybridization analysis. In embodiments, the method provided herein includes analytical strategies to decode and quantify the activities of candidate cis regulatory elements (e g., enhancers) directly within a cell and / or a tissue. In embodiments, the method provided herein enables the detection of transcriptional activity of a cis regulatory element (e.g., an enhancer) within its native cell environment and / or tissue environment. In embodiments, the method provided herein enables the detection of transcriptional activity of a cis regulatory element (e.g., an enhancer) within its native physiological context.

[0102] In embodiments, the DNA sequence has a protein encoding sequence, an enhancer sequence and a barcode sequence associated with the enhancer sequence. In embodiments, the DNA sequence further includes a promoter. In embodiments, the promoter is a minimal promoter. In embodiments, the minimal promoter is a cytomegalovirus (CMV) minimal promoter. In embodiments, the expression of the DNA sequence is dependent on the transcriptional activity of the enhancer. In embodiments, the enhancer sequence is a putative enhancer sequence. In embodiments, the enhancer sequence is designed using molecular cloning techniques well known in the art. In embodiments, the DNA sequence is cloned into a plasmid using molecular cloning techniques well known in the art. In embodiments, an exemplary' method for cloning the DNA sequence into a plasmid is described in detail in the examples section and in FIG. 2.

[0103] In embodiments, the plasmid is delivered to the cell through viral transfection, calcium phosphate transfection, liposomal transfection, nucleofection. sonoporation, transfection through heat shock, magnetifection, or electroporation. In embodiments, the plasmid is delivered to the cell through viral transfection. In embodiments, the plasmid is delivered to the cell through calcium phosphate transfection. In embodiments, the plasmid is delivered to the cell through liposomal transfection. In embodiments, the plasmid is delivered to the cell through nucleofection. In embodiments, the plasmid is delivered to the cell through sonoporation. Inembodiments, the plasmid is delivered to the cell through transfection through heat shock. In embodiments, the plasmid is delivered to the cell through magnetifection. In embodiments, the plasmid is delivered to the cell through electroporation.

[0104] In embodiments, the viral transfection includes cloning the plasmid into a viral vector. In embodiments, the viral vector is an Adeno-associated virus (AAV) vector. In embodiments the AAV vector is an AAV -PHP. eB vector. In embodiments the viral vector including the plasmid is administered to a subject comprising the cell. In embodiments, allowing the cell to transcribe the plasmid occurs for between about 1 day to about 8 weeks. In embodiments, allowing the cell to transcribe the plasmid occurs for between about 2 days to about 8 weeks. In embodiments, allowing the cell to transcribe the plasmid occurs for between about 3 days to about 8 weeks. In embodiments, allowing the cell to transcribe the plasmid occurs for between about 4 days to about 8 weeks. In embodiments, allowing the cell to transcribe the plasmid occurs for between about 5 days to about 8 weeks. In embodiments, allowing the cell to transcribe the plasmid occurs for between about 6 days to about 8 weeks. In embodiments, allowing the cell to transcribe the plasmid occurs for between about 1 week to about 8 weeks. In embodiments, allowing the cell to transcribe the plasmid occurs for between about 2 weeks to about 8 w eeks. In embodiments, allowing the cell to transcribe the plasmid occurs for betw een about 3 weeks to about 8 w eeks. In embodiments, allowing the cell to transcribe the plasmid occurs for between about 4 weeks to about 8 weeks. In embodiments, allowing the cell to transcribe the plasmid occurs for between about 5 weeks to about 8 weeks. In embodiments, allowing the cell to transcribe the plasmid occurs for between about 6 w eeks to about 8 weeks. In embodiments, allowing the cell to transcribe the plasmid occurs for betw een about 7 weeks to about 8 weeks. In embodiments, allowing the cell to transcribe the plasmid occurs for about 4 weeks.

[0105] In embodiments, the cell is fixed by crosslinking biomolecules within the cell. In embodiments, the fixing includes formaldehyde or paraformaldehyde. In embodiments, the fixing includes formaldehyde. In embodiments, the fixing includes paraformaldehyde. In embodiments, the fixing is light fixation. In embodiments, the light fixation includes contacting the cell with methanol and acetic acid. In embodiments, the fixing includes contacting the cell with methanol and acetic acid.

[0106] In embodiments, each of the different encoding probes in the plurality of different encoding probes is capable of hybridizing to a subsequence within the barcoded RNA transcript. In embodiments, each of the different encoding probes in the plurality of different encoding is capable of hybridizing to a different barcode subsequence within the barcode sequence. In embodiments, each encoding probe in the plurality of encoding probes includes a barcodebinding sequence and an adapter-binding sequence. In embodiments, the barcode-binding sequence is capable of hybridizing to a complementary barcode subsequence within the barcoded RNA transcript.

[0107] In embodiments, each of the encoding probe RNA transcript hybridized complexes includes an encoding probe hybridized to a barcoded RNA transcript. In embodiments, each of the encoding probe RNA transcript hybridized complexes includes an encoding probe hybridized to a barcode subsequence within a barcoded RNA transcript. In embodiments, each of the encoding probes is hybridized to a different barcode subsequence.

[0108] In embodiments, the barcoded RNA transcript is incubated with the plurality7of different encoding probes at 37 °C overnight. In embodiments, the barcoded RNA transcript is incubated with the plurality- of different encoding probes at 37 °C for between about 6 hours and about 18 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality of different encoding probes at 37 °C for between about 7 hours and about 18 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality' of different encoding probes at 37 °C for between about 8 hours and about 18 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality of different encoding probes at 37 °C for between about 9 hours and about 18 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality of different encoding probes at 37 °C for between about 10 hours and about 18 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality of different encoding probes at 37 °C for between about 11 hours and about 18 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality of different encoding probes at 37 °C for between about 12 hours and about 18 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality' of different encoding probes at 37 °C for between about 13 hours and about 18 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality of different encoding probes at 37 °C for between about 14 hours and about 18 hours. Inembodiments, the barcoded RNA transcript is incubated with the plurality’ of different encoding probes at 37 °C for between about 15 hours and about 18 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality of different encoding probes at 37 °C for betw een about 16 hours and about 18 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality of different encoding probes at 37 °C for between about 17 hours and about 18 hours.

[0109] In embodiments, the barcoded RNA transcript is incubated with the plurality of different encoding probes at 37 °C for between about 6 hours and about 17 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality' of different encoding probes at 37 °C for between about 6 hours and about 16 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality of different encoding probes at 37 °C for between about 6 hours and about 15 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality7of different encoding probes at 37 °C for betw een about 6 hours and about 14 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality of different encoding probes at 37 °C for between about 6 hours and about 13 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality of different encoding probes at 37 °C for between about 6 hours and about 12 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality' of different encoding probes at 37 °C for betw een about 6 hours and about 11 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality of different encoding probes at 37 °C for between about 6 hours and about 10 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality' of different encoding probes at 37 °C for between about 6 hours and about 9 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality of different encoding probes at 37 °C for between about 6 hours and about 8 hours. In embodiments, the barcoded RNA transcript is incubated with the plurality of different encoding probes at 37 °C for between about 6 hours and about 7 hours.

[0110] In embodiments, the barcoded RNA transcript is incubated with the plurality of different encoding probes in a buffer including formamide, saline-sodium citrate (SSC), a surfactant (e.g., a detergent), a polysaccharide (e.g., a dextran), and / or an RNase inhibitor. In embodiments, the RNA transcript is incubated with the plurality of different encoding probes ina buffer including 50% formamide, 2X saline-sodium citrate (SSC). 0. 1% Tween, 10% dextran sulfate, and an RNase inhibitor.

[0111] In embodiments, each different adapter probe in the first plurality of different adapter probes is capable of hybridizing to an encoding probe bound to the barcoded RNA transcript. In embodiments, each different adapter probe in the first plurality of different adapter probes is capable of hybridizing to a subsequence within an encoding probe. In embodiments, each different adapter probe in the first plurality of different adapter probes is capable of hybridizing to an adapter probe-binding sequence within an encoding probe. In embodiments, each of the adapter probes is hybridized to a different adapter probe-binding sequence.

[0112] In embodiments, each of the adapter probe RNA transcript hybridized complexes includes an adapter probe hybridized to an encoding probe RNA transcript hybridized complex. In embodiments, each of the adapter probe RNA transcript hybridized complexes includes an adapter probe hybridized to the encoding probe bound to the barcoded RNA transcript. In embodiments, each of the adapter probe RNA transcript hybridized complexes includes an adapter probe hybridized to an adapter probe-binding sequence within an encoding probe. In embodiments, each of the adapter probes is hybridized to a different encoding probe. In embodiments, each of the adapter probes is hybridized to a different adapter probe-binding sequence.

[0113] In embodiments, the plurality of encoding probe RNA transcript hybridized complexes is incubated with the plurality of different adapter probes in a buffer including formamide, saline-sodium citrate (SSC), and / or a surfactant (e.g., a detergent). In embodiments, the plurality of encoding probe RNA transcript hybridized complexes is incubated with the plurality of different adapter probes in a buffer including 35% formamide, 2X saline-sodium citrate (SSC), and 0.1% Tween. In embodiments, the plurality of encoding probe RNA transcript hybridized complexes is incubated with the plurality of different adapter probes for between about 30 minutes to about 3 hours. In embodiments, the plurality of encoding probe RNA transcript hybridized complexes is incubated with the plurality' of different adapter probes for between about 45 minutes to about 3 hours. In embodiments, the plurality' of encoding probe RNA transcript hybridized complexes is incubated with the plurality of different adapter probes for between about 1 hour to about 3 hours. In embodiments, the plurality of encoding probe RNAtranscript hybridized complexes is incubated with the plurality of different adapter probes for between about 1.5 hours to about 3 hours. In embodiments, the plurality of encoding probe RNA transcript hybridized complexes is incubated with the plurality of different adapter probes for between about 2 hours to about 3 hours. In embodiments, the plurality of encoding probe RNA transcript hybridized complexes is incubated with the plurality of different adapter probes for between about 2.5 hours to about 3 hours. In embodiments, the plurality’ of encoding probe RNA transcript hybridized complexes is incubated with the plurality of different adapter probes for about 1.5 hours.

[0114] In embodiments, follow ing hybridization, the plurality of adapter probe RNA transcript hybridized complexes is washed with a buffer including formamide, saline-sodium citrate (SSC), and / or a surfactant (e.g., a detergent). In embodiments, following hybridization, the plurality of adapter probe RNA transcript hybridized complexes is washed with a buffer including 30% formamide, 2X saline-sodium citrate (SSC), and 0.1% Tween.

[0115] In embodiments, each of the different fluorescent readout probes w ithin the plurality of different fluorescent readout probes is capable of hybridizing to an adapter probe bound to an encoding probe bound to the barcoded RNA transcript. In embodiments, each different fluorescent readout probe in the plurality’ of different fluorescent readout probes is capable of hybridizing to a subsequence w ithin an adapter probe. In embodiments, each different fluorescent readout in the plurality of different fluorescent readout probes is capable of hybridizing to a fluorescent readout probe-binding sequence within an adapter probe. In embodiments, each of the fluorescent readout probes is hybridized to a different fluorescent readout probe-binding sequence.

[0116] In embodiments, each of the fluorescent readout probe RNA transcript hybridized complexes includes a fluorescent readout probe hy bridized to an adapter probe RNA transcript hybridized complex. In embodiments, each of the fluorescent readout probe RNA transcript hybridized complexes includes a fluorescent readout probe hybridized to the adapter probe bound to the encoding probe bound to the barcoded RNA transcript. In embodiments, each of the fluorescent readout probe RNA transcript hybridized complexes includes a fluorescent readout probe hybridized to a fluorescent readout probe-binding sequence within an adapter probe. In embodiments, each of the fluorescent readout probes is hybridized to a different adapter probe.In embodiments, each of the fluorescent readout probes is hybridized to a different fluorescent readout probe-binding sequence.

[0117] In embodiments, the plurality of adapter probe RNA transcript hybridized complexes is incubated with the plurality of different fluorescent readout probes in a buffer including formamide, saline-sodium citrate (SSC), and / or a surfactant (e.g., a detergent). In embodiments, the plurality of adapter probe RNA transcript hybridized complexes is incubated with the plurality of different fluorescent readout probes in a buffer including 35% formamide, 2X saline- sodium citrate (SSC), and 0.1% Tween. In embodiments, the plurality' of adapter probe RNA transcript hybridized complexes is incubated with the plurality of different fluorescent readout probes for between about 15 minutes to about 2 hours. In embodiments, the plurality of adapter probe RNA transcript hybridized complexes is incubated with the plurality of different fluorescent readout probes for between about 20 minutes to about 2 hours. In embodiments, the plurality of adapter probe RNA transcript hybridized complexes is incubated with the plurality of different fluorescent readout probes for between about 30 minutes to about 2 hours. In embodiments, the plurality of adapter probe RNA transcript hybridized complexes is incubated with the plurality of different fluorescent readout probes for between about 45 minutes to about 2 hours. In embodiments, the plurality of adapter probe RNA transcript hybridized complexes is incubated with the plurality of different fluorescent readout probes for between about 1 hour to about 2 hours. In embodiments, the plurality of adapter probe RNA transcript hybridized complexes is incubated with the plurality of different fluorescent readout probes for between about 1.5 hours to about 2 hours. In embodiments, the plurality of adapter probe RNA transcript hybridized complexes is incubated with the plurality' of different fluorescent readout probes for about 30 minutes.

[0118] In embodiments, following hybridization, the plurality of fluorescent readout probe RNA transcript hybridized complexes is washed with a buffer including formamide, saline- sodium citrate (SSC), and / or a surfactant (e g., a detergent). In embodiments, following hybridization, the plurality of fluorescent readout probe RNA transcript hybridized complexes is washed with a buffer including 30% formamide. 2X saline-sodium citrate (SSC), and 0.1% Tween.

[0119] In embodiments, the first plurality of different fluorescent readout probes hybridized to the barcoded RNA transcript is detected using fluorescent microscopy. In embodiments, the plurality of different fluorescent readout probes are detected using fluorescent microscopy.

[0120] In embodiments, the method further includes denaturing the hybridized adapter probes and the hybridized fluorescent readout probes from the plurality of encoding probe RNA transcript hybridized complexes, thereby producing a plurality’ of free adapter probes and a plurality of free fluorescent readout probes. In embodiments, the denaturing includes contacting each of the adapter probe RNA transcript hybridized complexes with a probe-stripping buffer. In embodiments, the denaturing includes washing each of the adapter probe RNA transcript hybridized complexes with a probe-stripping buffer. In embodiments, the denaturing includes contacting each of the fluorescent readout probe RNA transcript hybridized complexes with a probe-stripping buffer. In embodiments, the denaturing includes washing each of the fluorescent readout probe RNA transcript hybridized complexes with a probe-stripping buffer. In embodiments, the probe-stripping buffer includes formamide and / or saline-sodium citrate (SSC). In embodiments, the probe-stripping buffer includes formamide and saline-sodium citrate (SSC). In embodiments, the probe-stripping buffer includes about 80% formamide and about 0.8X SSC. In embodiments, the probe-stripping buffer includes formamide. In embodiments, the probestripping buffer includes about 80% formamide. In embodiments, the probe-stripping buffer includes saline-sodium citrate (SSC). In embodiments, the probe-stripping buffer includes about 0.8X SSC. In embodiments, the probe-stripping buffer does not denature the plurality of encoding probe RNA transcript complexes.

[0121] In embodiments, the method further includes separating the plurality of free adapter probes and the plurality of free fluorescent readout probes thereby providing a plurality of separated encoding probe RNA transcript hybridized complexes. In embodiments, the separating includes washing the plurality of free adapter probes and the plurality of free fluorescent readout probes from the plurality of the encoding probe RNA transcript hybridized complexes. In embodiments, the separating includes washing the plurality of free adapter probes from the plurality of the encoding probe RNA transcript hybridized complexes. In embodiments, the separating includes washing the plurality of free fluorescent readout probes from the plurality of the encoding probe RNA transcript hybridized complexes. In embodiments, the separatingincludes washing the plurality of free adapter probes and the plurality of free fluorescent readout probes from the hydrogel. In embodiments, the separating includes washing the plurality of free adapter probes from the hydrogel. In embodiments, the separating includes washing the plurality of free fluorescent readout probes from the hydrogel.

[0122] In embodiments, the method further includes contacting the plurality' of separated encoding probe RNA transcript hybridized complexes w ith a second plurality of different adapter probes and allowing each of the different adapter probe in the second plurality of different adapter probes to hybridize to a different encoding probe bound to the barcoded RNA transcript thereby forming a second plurality of adapter probe RNA transcript hybridized complexes. In embodiments, the second plurality of different adapter probes is different from the first plurality of different adapter probes.

[0123] In embodiments, the method further includes contacting the second plurality of adapter probe RNA transcript hybridized complexes with the plurality' of different fluorescent readout probes and allowing each of the different fluorescent readout probes to hybridize to a different adapter probe bound to the barcoded RNA transcript thereby forming a second plurality of fluorescent readout probe RNA transcript hybridized complexes.

[0124] In embodiments, the method further includes detecting the second plurality of different fluorescent readout probes hybridized to the barcoded RNA transcript, thereby detecting the barcoded RNA transcript and the enhancer transcriptional activity.

[0125] In embodiments, the method further includes following step (f): (g) denaturing the hybridized adapter probes and the hybridized fluorescent readout probes from the plurality of encoding probe RNA transcript hybridized complexes, thereby producing a plurality of free adapter probes and a plurality of free fluorescent readout probes; (h) separating the plurality of free adapter probes and the plurality of free fluorescent readout probes thereby providing a plurality of separated encoding probe RNA transcript hy bridized complexes; (i) contacting the plurality of separated encoding probe RNA transcript hybridized complexes with a second plurality of different adapter probes and allowing each of the different adapter probe in the second plurality of different adapter probes to hybridize to a different encoding probe bound to the barcoded RNA transcript thereby forming a second plurality of adapter probe RNA transcripthybridized complexes, wherein the second plurality of different adapter probes are different from the first plurality of different adapter probes; (j) contacting the second plurality of adapter probe RNA transcript hybridized complexes with the plurality of different fluorescent readout probes and allowing each of the different fluorescent readout probes to hybridize to a different adapter probe bound to the barcoded RNA transcript thereby forming a second plurality of fluorescent readout probe RNA transcript hybridized complexes; (k) detecting the second plurality of different fluorescent readout probes hybridized to the barcoded RNA transcript, thereby detecting the barcoded RNA transcript and the enhancer transcriptional activity.

[0126] In embodiments, the method further includes repeating steps (g)-(k). In embodiments, the method includes repeating steps (g)-(k) at least 1 time. In embodiments, the method includes repeating steps (g)-(k) at least 2 times. In embodiments, the method includes repeating steps (g)- (k) at least 3 times. In embodiments, the method includes repeating steps (g)-(k) at least 4 times. In embodiments, the method includes repeating steps (g)-(k) at least 5 times. In embodiments, the method includes repeating steps (g)-(k) at least 6 times. In embodiments, the method includes repeating steps (g)-(k) at least 7 times. In embodiments, the method includes repeating steps (g)- (k) at least 8 times. In embodiments, the method includes repeating steps (g)-(k) at least 9 times. In embodiments, the method includes repeating steps (g)-(k) at least 10 times. In embodiments, the method includes repeating steps (g)-(k) at least 11 times. In embodiments, the method includes repeating steps (g)-(k) at least 12 times. In embodiments, the method includes repeating steps (g)-(k) at least 13 times. In embodiments, the method includes repeating steps (g)-(k) at least 14 times. In embodiments, the method includes repeating steps (g)-(k) at least 15 times. In embodiments, the method includes repeating steps (g)-(k) at least 16 times.

[0127] In embodiments, the method includes repeating steps (g)-(k) 1 time. In embodiments, the method includes repeating steps (g)-(k) 2 times. In embodiments, the method includes repeating steps (g)-(k) 3 times. In embodiments, the method includes repeating steps (g)-(k) 4 times. In embodiments, the method includes repeating steps (g)-(k) 5 times. In embodiments, the method includes repeating steps (g)-(k) 6 times. In embodiments, the method includes repeating steps (g)-(k) 7 times. In embodiments, the method includes repeating steps (g)-(k) 8 times. In embodiments, the method includes repeating steps (g)-(k) 9 times. In embodiments, the method includes repeating steps (g)-(k) 10 times. In embodiments, the method includes repeating steps(g)-(k) 11 times. In embodiments, the method includes repeating steps (g)-(k) 12 times. In embodiments, the method includes repeating steps (g)-(k) 13 times. In embodiments, the method includes repeating steps (g)-(k) 14 times. In embodiments, the method includes repeating steps (g)-(k) 15 times. In embodiments, the method includes repeating steps (g)-(k) 16 times.

[0128] In embodiments, each repeated step (i) (e.g., second, third, fourth, fifth, sixth etc.) includes contacting the plurality of encoding probe RNA transcript hybridized complexes with a corresponding (e.g., second, third, fourth fifth, sixth, etc.) plurality of different adapter probes, wherein the corresponding plurality of different probes used in each repeated step (i) is different. In embodiments, the method further includes allowing each of the corresponding plurality of different adapter probe in each repeated step to hybridize to a different encoding probe bound to the barcoded RNA transcript thereby forming a corresponding plurality of adapter probe RNA transcript hybridized complexes. In embodiments, each repeated step (i) (e.g., second, third, fourth, fifth, sixth etc.) includes contacting the plurality of encoding probe RNA transcript hybridized complexes with a corresponding (e g., second, third, fourth fifth, sixth, etc.) plurality of different adapter probes, wherein the corresponding plurality of different probes used in each repeated step (i) is different, and allowing each of the corresponding plurality of different adapter probes in each repeated step to hybridize to a different encoding probe bound to the barcoded RNA transcript thereby forming a corresponding plurality of adapter probe RNA transcript hybridized complexes.

[0129] In embodiments, each repeated step (j) (e.g., second, third, fourth, fifth, sixth etc.) includes contacting the corresponding plurality of adapter probe RNA transcript hybridized complexes with the plurality of different fluorescent readout probes. In embodiments, the method further includes allowing each of the different fluorescent readout probes in each repeated step (j) to hybridize to a different adapter probe bound to the barcoded RNA transcript thereby forming a corresponding plurality of fluorescent readout probe RNA transcript hybridized complexes. In embodiments, each repeated step (j) (e g., second, third, fourth, fifth, sixth etc.) includes contacting the corresponding plurality of adapter probe RNA transcript hybridized complexes with the plurality of different fluorescent readout probes and allowing each of the different fluorescent readout probes in each repeated step (j) to hybridize to a different adapter probe bound to the barcoded RNA transcript thereby forming a correspondingplurality of fluorescent readout probe RNA transcript hybridized complexes. In embodiments, the plurality of fluorescent readout probes in each repeated step (j) is the same.

[0130] In embodiments, each repeated step (k) (e g., second, third, fourth, fifth, sixth etc.)includes detecting the corresponding (e.g., second, third, fourth, fifth, sixth etc.) plurality' of different fluorescent readout probes hybridized to the barcoded RNA transcript, thereby detecting the barcoded RNA transcript and the enhancer transcriptional activity

[0131] In embodiments, each encoding probe in the plurality of encoding probes includes a barcode-binding sequence and an adapter-binding sequence, wherein the barcode-binding sequence is capable of hybridizing to a complementary' barcode subsequence within the barcoded RNA transcript. In embodiments, each encoding probe in the plurality of encoding probes includes a barcode-binding sequence and an adapter-binding sequence. In embodiments, the barcode-binding sequence is capable of hybridizing to a complementary barcode subsequence within the barcoded RNA transcript. In embodiments, the encoding probe includes an acrydite modification on the 5’ end. In embodiments, the encoding probe is an acrydite-modified encoding probe. In embodiments, the acrydite-modified encoding probe is an encoding probe with an acrydite modification on the 5' end. In embodiments, the acrydite modified-encoding probe is capable of co-polymerization into an acrylamide gel.

[0132] In embodiments, each adapter probe in the first plurality', second plurality, corresponding plurality' of adapter probes includes an encoding probe-binding sequence wherein the encoding probe-binding sequence is capable of binding to a complementary adapter-binding sequence within an encoding probe; and a fluorescent readout probe-binding sequence. In embodiments, each adapter probe in the first plurality, second plurality, corresponding plurality of adapter probes includes an encoding probe-binding sequence. In embodiments, the encoding probe-binding sequence is capable of binding to a complementary adapter-binding sequence within an encoding probe. In embodiments, the first plurality, second plurality, corresponding plurality of adapter probes includes a fluorescent readout probe-binding sequence.

[0133] In embodiments, each fluorescent readout probe in the plurality of the fluorescent readout probes includes a complementary' fluorescent readout probe-binding sequence and a chemical fluorophore. In embodiments, the fluorescent readout probe is capable of hybridizing toan adapter probe RNA transcript complex. In embodiments, the fluorescent readout probe is capable of hybridizing to an adapter probe. In embodiments, each fluorescent readout probe in the plurality of fluorescent readout probes is capable of hybridizing to the fluorescent readout probe-binding sequence within an adapter probe. In embodiments, the fluorescent readout probe is capable of hybridizing to a fluorescent readout probe-binding sequence within the adapter probe. In embodiments, the complementary fluorescent readout probe-binding sequence is capable of hybridizing to a fluorescent readout probe binding sequence within the adapter probe. In embodiments, the fluorescent readout probe is capable of hybridizing to an adapter probe RNA transcript complex.

[0134] In embodiments, the chemical fluorophore is an Alexa Fluor 750 fluorophore. a Cy5 fluorophore, a Cy3 fluorophore, a 6-FAM fluorophore, a 6-FAM fluorophore, a Fluorescein dT fluorophore, a TAMRA fluorophore, a JOE fluorophore, a MAX fluorophore, a TET fluorophore, a TET fluorophore, a Cy5.5 fluorophore, a ROX fluorophore, a TYE 563 fluorophore, a Yakima Yellow fluorophore, a HEX fluorophore, a TEX 615 fluorophore, a TYE 665 fluorophore, a TYE 705 fluorophore, a SUN fluorophore. an ATTO 425 fluorophore, an ATTO 488 fluorophore, an ATTO 532 fluorophore, an ATTO 550 fluorophore, an ATTO 565 fluorophore, an ATTO RholOl fluorophore, an ATTO 590 fluorophore, an ATTO 633 fluorophore, an ATTO 647N fluorophore, an ATTO 700 fluorophore, an Alex Fluor 405 fluorophore, an Alex Fluor 488 fluorophore, an Alex Fluor 532 fluorophore, an Alex Fluor 546 fluorophore, an Alex Fluor 594 fluorophore. an Alex Fluor 647 fluorophore, an Alex Fluor 660 fluorophore, a 5' IRDye 700 fluorophore, a 5' IRDye 800 fluorophore, a 5' IRDye 800CW fluorophore, a Rhodamine Green-X fluorophore, a Rhodamine Red fluorophore, a 5-TAMRA fluorophore, a Texas Red-X fluorophore, a Lightcycler 640 fluorophore, or a Dy 750 fluorophore. In embodiments, the chemical fluorophore is an Alexa Fluor 750 fluorophore. In embodiments, the chemical fluorophore is a Cy5 fluorophore. In embodiments, the chemical fluorophore is a Cy3 fluorophore. In embodiments, the chemical fluorophore is a 6-FAM fluorophore. In embodiments, the chemical fluorophore is a 6-FAM fluorophore. In embodiments, the chemical fluorophore is a Fluorescein dT fluorophore. In embodiments, the chemical fluorophore is a TAMRA fluorophore. In embodiments, the chemical fluorophore is a JOE fluorophore. In embodiments, the chemical fluorophore is a MAX fluorophore. Inembodiments, the chemical fluorophore is a TET fluorophore. In embodiments, the chemical fluorophore is a TET fluorophore. In embodiments, the chemical fluorophore is a Cy5.5 fluorophore. In embodiments, the chemical fluorophore is a ROX fluorophore. In embodiments, the chemical fluorophore is a TYE 563 fluorophore. In embodiments, the chemical fluorophore is a Yakima Yellow fluorophore. In embodiments, the chemical fluorophore is a HEX fluorophore. In embodiments, the chemical fluorophore is a TEX 615 fluorophore. In embodiments, the chemical fluorophore is a TYE 665 fluorophore. In embodiments, the chemical fluorophore is a TYE 705 fluorophore. In embodiments, the chemical fluorophore is a SUN fluorophore. In embodiments, the chemical fluorophore is an ATTO 425 fluorophore. In embodiments, the chemical fluorophore is an ATTO 488 fluorophore. In embodiments, the chemical fluorophore is an ATTO 532 fluorophore. In embodiments, the chemical fluorophore is an ATTO 550 fluorophore. In embodiments, the chemical fluorophore is an ATTO 565 fluorophore. In embodiments, the chemical fluorophore is an ATTO RholOl fluorophore. In embodiments, the chemical fluorophore is an ATTO 590 fluorophore. In embodiments, the chemical fluorophore is an ATTO 633 fluorophore. In embodiments, the chemical fluorophore is an ATTO 647N fluorophore. In embodiments, the chemical fluorophore is an ATTO 700 fluorophore. In embodiments, the chemical fluorophore is an Alex Fluor 405 fluorophore. In embodiments, the chemical fluorophore is an Alex Fluor 488 fluorophore. In embodiments, the chemical fluorophore is an Alex Fluor 532 fluorophore. In embodiments, the chemical fluorophore is an Alex Fluor 546 fluorophore. In embodiments, the chemical fluorophore is an Alex Fluor 594 fluorophore. In embodiments, the chemical fluorophore is an Alex Fluor 647 fluorophore. In embodiments, the chemical fluorophore is an Alex Fluor 660 fluorophore. In embodiments, the chemical fluorophore is a 5' IRDye 700 fluorophore. In embodiments, the chemical fluorophore is a 5' IRDye 800 fluorophore. In embodiments, the chemical fluorophore is a 5' IRDye 800CW fluorophore. In embodiments, the chemical fluorophore is a Rhodamine Green-X fluorophore. In embodiments, the chemical fluorophore is a Rhodamine Red fluorophore. In embodiments, the chemical fluorophore is a 5-TAMRA fluorophore. In embodiments, the chemical fluorophore is a Texas Red-X fluorophore. In embodiments, the chemical fluorophore is a Lightcycler 640 fluorophore. In embodiments, the chemical fluorophore is a Dy 750 fluorophore. In embodiments, the chemical fluorophore is selected from an Alexa 750 fluorophore, a Cy5 fluorophore, or a Cy3 fluorophore. In embodiments, thechemical fluorophore is selected from the group consisting of an Alexa 750 fluorophore, a Cy5 fluorophore, and a Cy3 fluorophore. In embodiments, the plurality of fluorescent readout probes includes a plurality of Alexa 750 fluorophores, a plurality of Cy 5 fluorophores, and / or a plurality' of Cy3 fluorophores. In embodiments, the plurality of fluorescent readout probes includes a plurality of Alexa 750 fluorophores, a plurality of Cy5 fluorophores, and a plurality of Cy3 fluorophores. In embodiments, the plurality of fluorescent readout probes includes a plurality of Alexa 750 fluorophores, a plurality’ of Cy5 fluorophores, or a plurality of Cy3 fluorophores. In embodiments, the plurality of fluorescent readout probes includes a plurality of Alexa 750 fluorophores. In embodiments, the plurality of fluorescent readout probes includes a plurality of Cy5 fluorophores. In embodiments, the plurality of fluorescent readout probes includes a plurality of Cy3 fluorophores.

[0135] In embodiments, the cell that is fixed forms part of a tissue including the cell. In embodiments, the method includes a plurality of fixed cells in a tissue.

[0136] In embodiments, following step (c) and prior to step (d), the method further includes transferring the plurality of encoding probe RNA transcript hybridized complexes to a hydrogel. In embodiments, the plurality of encoding probe RNA transcript hybridized complexes are embedded in a hydrogel. In embodiments, the hydrogel is an acrylamide gel. In embodiments, the hydrogel is a 4% acry lamide gel. In embodiments, the hydrogel is a polyacrylamide gel. In embodiments, the hydrogel is a 4% polyacrylamide gel. In embodiments, prior to transferring the plurality of encoding probe RNA transcript hybridized complexes to the hydrogel, the plurality of encoding probe RNA transcript hybridized complexes are washed in the pre-hybridization buffer.

[0137] In embodiments, following step (c) and prior to step (d), the method further includes transferring the plurality of encoding probe antisense barcode transcript hybridized complexes to a hydrogel. In embodiments, the plurality of encoding probe antisense barcode transcript hybridized complexes are embedded in a hydrogel. In embodiments, the hydrogel is an acrylamide gel. In embodiments, the hydrogel is a 4% acrylamide gel. In embodiments, the hydrogel is a polyacry lamide gel. In embodiments, the hydrogel is a 4% polyacrylamide gel. In embodiments, prior to transferring the plurality of encoding probe antisense barcode transcripthybridized complexes to the hydrogel, the plurality of encoding probe antisense barcode transcript hybridized complexes are washed in the pre-hybridization buffer.

[0138] In embodiments, following the transferring the plurality of encoding probe RNA transcript hybridized complexes to the hydrogel, the plurality of encoding probe RNA transcript hybridized complexes are post-fixed with paraformaldehyde and saline-sodium citrate (SSC). In embodiments, the post-fixing includes 4% paraformaldehyde and 2X SSC.

[0139] In embodiments, following the transferring the plurality of encoding probe antisense barcode transcript hybridized complexes to the hydrogel, the plurality of encoding probe antisense barcode transcript hybridized complexes are post-fixed wi th paraformaldehyde and saline-sodium citrate (SSC). In embodiments, the post-fixing includes 4% paraformaldehyde and 2X SSC.

[0140] In embodiments, the hydrogel is incubated in a tissue clearing solution at 37 °C for between about 24 hours to about 48 hours. In embodiments, the tissue clearing solution includes 2.5% SDS, 0.5% Triton X-100, and 1% Proteinase K in 2X saline-sodium citrate (SSC). In embodiments, following the tissue clearing solution incubation and prior to imaging, the hydrogel is washed in 2X saline-sodium citrate (SSC).

[0141] In embodiments, the DNA sequence includes from 5' to 3': the protein encoding sequence, the enhancer sequence, and the barcode sequence.

[0142] In embodiments, the DNA sequence further includes a T7 polymerase promoter sequence 3' of the barcode sequence. In embodiments, following step (b) and prior to step (c) the method further includes contacting the DNA sequence with a T7 polymerase, thereby transcribing an antisense barcode transcript. In embodiments, the fixing of step (b) includes light fixation including methanol and acetic acid. In embodiments, following fixing the cell with methanol and acetic acid of step (b) and prior to step (c) the method further includes contacting the DNA sequence with a T7 polymerase, thereby transcribing an antisense barcode transcript. In embodiments, the T7 polymerase and DNA sequence are incubated for between about 3 hours and about 16 hours. In embodiments, the T7 polymerase and the DNA sequence are incubated together at about 40 °C.

[0143] In embodiments, following the transcription of the antisense barcode transcript, the cell is fixed in paraformaldehyde. In embodiments, following the transcription of the antisense barcode transcript, the cell is fixed in 4% paraformaldehyde. In embodiments, following the paraformaldehyde fixation, the cell is permeabilized. In embodiments, following the paraformaldehyde fixation, the cell is permeabilized with Triton X-100. In embodiments. following the paraformaldehyde fixation, the cell is permeabilized with 0.05% Triton X-100. In embodiments, following the permeabilization, the cell is contacted with ethanol. In embodiments, following the permeabilization, the cell is contacted with 80% ethanol.

[0144] In embodiments, prior to step (c), the cell is incubated in a pre-hybridization buffer. In embodiments, the pre-hybridization buffer includes formamide, saline-sodium citrate (SSC), a surfactant (e.g., detergent), and / or RNase inhibitor. In embodiments, the pre-hybridization buffer includes 40% formamide, 2X saline-sodium citrate (SSC), 0.1% Tween, and RNase inhibitor.

[0145] In embodiments, step (c) further includes contacting the antisense barcode transcript with a plurality of different antisense encoding probes and allowing each of the different antisense encoding probes to hybridize to a different subsequence within the antisense barcode transcript thereby forming a plurality of encoding probe antisense barcode transcript hybridized complexes. In embodiments, step (c) includes contacting the antisense barcode transcript with a plurality7of different antisense encoding probes. In embodiments, step (c) includes alloyving each of the different antisense encoding probes to hybridize to a different subsequence w ithin the antisense barcode transcript thereby forming a plurality of encoding probe antisense barcode transcript hybridized complexes.

[0146] In embodiments, each of the different antisense encoding probes in the plurality of different antisense encoding probes is capable of hybridizing to a subsequence within the antisense barcode transcript. In embodiments, each of the different antisense encoding probes in the plurality of different antisense encoding probes is capable of hybridizing to a different antisense barcode subsequence within the antisense barcode transcript. In embodiments, each antisense encoding probe in the plurality of antisense encoding probes includes an antisense barcode-binding sequence and an antisense adapter-binding sequence. In embodiments, theantisense barcode-binding sequence is capable of hybridizing to a complementary antisense barcode subsequence within the antisense barcode transcript.

[0147] In embodiments, each of the encoding probe antisense barcode transcript hybridized complexes includes an antisense encoding probe hybridized to an antisense barcode transcript. In embodiments, each of the encoding probe antisense barcode transcript hybridized complexes includes an antisense encoding probe hybridized to an antisense barcode subsequence within an antisense barcode transcript. In embodiments, each of the antisense encoding probes is hybridized to a different antisense barcode subsequence.

[0148] In embodiments, step (d) further includes contacting the plurality of encoding probe antisense barcode transcript hybridized complexes with a first plurality of different antisense adapter probes and allowing each of the different antisense adapter probes to hybridize to a different antisense encoding probe bound to the antisense barcode transcript thereby forming a first plurality of adapter probe antisense barcode transcript hybridized complexes. In embodiments, step (d) includes contacting the plurality of encoding probe antisense barcode transcript hybridized complexes with a first plurality of different antisense adapter probes. In embodiments, step (d) includes allowing each of the different antisense adapter probes to hybridize to a different antisense encoding probe bound to the antisense barcode transcript thereby forming a first plurality of adapter probe antisense barcode transcript hybridized complexes.

[0149] In embodiments, each different antisense adapter probe in the first plurality of different antisense adapter probes is capable of hybridizing to an antisense encoding probe bound to the antisense barcode transcript. In embodiments, each different antisense adapter probe in the first plurality of different antisense adapter probes is capable of hybridizing to a subsequence within an antisense encoding probe. In embodiments, each different antisense adapter probe in the first plurality of different antisense adapter probes is capable of hybridizing to an antisense adapter probe-binding sequence within an antisense encoding probe. In embodiments, each of the antisense adapter probes is hybridized to a different antisense adapter probe-binding sequence.

[0150] In embodiments, each of the adapter probe antisense barcode transcript hybridized complexes includes an antisense adapter probe hybridized to an encoding probe antisensebarcode transcript hybridized complex. In embodiments, each of the adapter probe antisense barcode transcript hybridized complexes includes an antisense adapter probe hybridized to the antisense encoding probe bound to the antisense barcode transcript. In embodiments, each of the adapter probe antisense barcode transcript hybridized complexes includes an antisense adapter probe hybridized to an antisense adapter probe-binding sequence within an antisense encoding probe. In embodiments, each of the antisense adapter probes is hybridized to a different antisense encoding probe. In embodiments, each of the antisense adapter probes is hybridized to a different antisense adapter probe-binding sequence.

[0151] In embodiments, step (e) further includes contacting the first plurality of adapter probe antisense barcode transcript hybridized complexes with a first plurality of different fluorescent readout probes and allowing each of the different fluorescent readout probes to hybridize to a different adapter probe bound to the antisense barcode transcript thereby forming a first plurality of fluorescent readout probe antisense barcode transcript hybridized complexes. In embodiments, step (e) includes contacting the first plurality of adapter probe antisense barcode transcript hybridized complexes with a first plurality of different fluorescent readout probes. In embodiments, step (e) includes allowing each of the different fluorescent readout probes to hybridize to a different adapter probe bound to the antisense barcode transcript thereby forming a first plurality of fluorescent readout probe antisense barcode transcript hybridized complexes.

[0152] In embodiments, each of the different fluorescent readout probes within the plurality of different fluorescent readout probes is capable of hybridizing to an antisense adapter probe bound to an antisense encoding probe bound to the barcoded RNA transcript. In embodiments, each different fluorescent readout probe in the plurality of different fluorescent readout probes is capable of hybridizing to a subsequence within an antisense adapter probe. In embodiments, each different fluorescent readout probe in the plurality of different fluorescent readout probes is capable of hybridizing to a fluorescent readout probe-binding sequence within an antisense adapter probe. In embodiments, each of the fluorescent readout probes is hybridized to a different fluorescent readout probe-binding sequence.

[0153] In embodiments, each of the fluorescent readout probe antisense barcode transcript hybridized complexes includes a fluorescent readout probe hybridized to an adapter probe antisense barcode transcript hybridized complex. In embodiments, each of the fluorescentreadout probe antisense barcode transcript hybridized complexes includes a fluorescent readout probe hybridized to the antisense adapter probe bound to the antisense encoding probe bound to the antisense barcode transcript. In embodiments, each of the fluorescent readout probe antisense barcode transcript hybridized complexes includes a fluorescent readout probe hybridized to a fluorescent readout probe-binding sequence within an antisense adapter probe. In embodiments, each of the fluorescent readout probes is hybridized to a different antisense adapter probe. In embodiments, each of the fluorescent readout probes is hybridized to a different fluorescent readout probe-binding sequence.

[0154] In embodiments, step (f) further includes detecting the first plurality of different fluorescent readout probes hybridized to the antisense barcode transcript thereby detecting the antisense barcode transcript.

[0155] In embodiments, the first plurality of different fluorescent readout probes hybridized to the barcoded RNA transcript is detected using fluorescent microscopy. In embodiments, the plurality7of different fluorescent readout probes are detected using fluorescent microscopy.

[0156] In embodiments, step (c) further includes contacting the antisense barcode transcript with a plurality of different antisense encoding probes and allowing each of the different antisense encoding probes to hybridize to a different subsequence within the antisense barcode transcript thereby forming a plurality of encoding probe antisense barcode transcript hybridized complexes; step (d) further includes contacting the plurality of encoding probe antisense barcode transcript hybridized complexes with a first plurality of different antisense adapter probes and allowing each of the different antisense adapter probes to hybridize to a different antisense encoding probe bound to the antisense barcode transcript thereby forming a first plurality of adapter probe antisense barcode transcript hybridized complexes; step (e) further includes contacting the first plurality of adapter probe antisense barcode transcript hybridized complexes with a first plurality of different fluorescent readout probes and allowing each of the different fluorescent readout probes to hybridize to a different adapter probe bound to the antisense barcode transcript thereby forming a first plurality of fluorescent readout probe antisense barcode transcript hybridized complexes; and step (f further comprises detecting the first plurality of different fluorescent readout probes hybridized to the antisense barcode transcript thereby detecting the antisense barcode transcript.

[0157] In embodiments, each antisense encoding probe in the plurality of antisense encoding probes includes an antisense barcode-binding sequence and an antisense adapter-binding sequence, wherein the antisense barcode-binding sequence is capable of hybridizing to a complementary antisense barcode subsequence within the antisense barcode transcript. In embodiments, each antisense encoding probe in the plurality of antisense encoding probes includes an antisense barcode-binding sequence and an antisense adapter-binding sequence. In embodiments, the antisense barcode-binding sequence is capable of hybridizing to a complementary' antisense barcode subsequence within the antisense barcode transcript. In embodiments, the antisense encoding probe includes an acrydite modification on the 5' end. In embodiments, the antisense encoding probe is an acr dite-modified antisense encoding probe. In embodiments, the acrydite-modified antisense encoding probe is an antisense encoding probe with an acrydite modification on the 5' end. In embodiments, the aery di te-modi fied antisense encoding probe is capable of polymerization into an acry lamide gel or a polyacrylamide gel.

[0158] In embodiments, each antisense adapter probe in the first plurality, second plurality, corresponding plurality of antisense adapter probes includes an antisense encoding probebinding sequence wherein the antisense encoding probe-binding sequence is capable of binding to a complementary' antisense adapter-binding sequence within an antisense encoding probe; and a fluorescent readout probe-binding sequence. In embodiments, each adapter probe in the first plurality, second plurality’, corresponding plurality of antisense adapter probes includes an antisense encoding probe-binding sequence. In embodiments, the antisense encoding probebinding sequence is capable of binding to a complementary antisense adapter-binding sequence within an antisense encoding probe. In embodiments, the first plurality', second plurality', corresponding plurality of antisense adapter probes includes a fluorescent readout probe-binding sequence.

[0159] In embodiments, each fluorescent readout probe in the plurality of fluorescent readout probes is capable of hybridizing to the fluorescent readout probe-binding sequence w ithin an antisense adapter probe.

[0160] In embodiments, step (g) includes denaturing the hybridized antisense adapter probes and the hybridized fluorescent readout probes from the plurality of encoding probe antisense barcode transcript hybridized complexes thereby producing the plurality of free antisense adapterprobes and the plurality of free fluorescent readout probes. In embodiments, the denaturing includes contacting each of the adapter probe antisense barcode transcript hybridized complexes with a probe-stripping buffer. In embodiments, the denaturing includes w ashing each of the adapter probe antisense barcode transcript hybridized complexes with a probe-stripping buffer. In embodiments, the denaturing includes contacting each of the fluorescent readout probe antisense barcode transcript hybridized complexes with a probe-stripping buffer. In embodiments, the denaturing includes w ashing each of the fluorescent readout probe antisense barcode transcript hybridized complexes with a probe-stripping buffer. In embodiments, the probe-stripping buffer includes formamide and / or saline-sodium citrate (SSC). In embodiments, the probe-stripping buffer includes formamide and saline-sodium citrate (SSC). In embodiments, the probe-stripping buffer includes about 80% formamide and about 0.8X SSC. In embodiments, the probe-stripping buffer includes formamide. In embodiments, the probe-stripping buffer includes about 80% formamide. In embodiments, the probe-stripping buffer includes saline- sodium citrate (SSC). In embodiments, the probe-stripping buffer includes about 0.8X SSC. In embodiments, the probe-stripping buffer does not denature the plurality of encoding probe antisense barcode transcript complexes.

[0161] In embodiments, the method further includes separating the plurality of free antisense adapter probes and the plurality' of free fluorescent readout probes thereby providing a plurality of separated encoding probe antisense barcode transcript hybridized complexes. In embodiments, the separating includes washing the plurality of free antisense adapter probes and the plurality of free fluorescent readout probes from the plurality of the encoding probe antisense barcode transcript hybridized complexes. In embodiments, the separating includes w ashing the plurality of free antisense adapter probes from the plurality of the encoding probe antisense barcode transcript hybridized complexes. In embodiments, the separating includes washing the plurality’ of free fluorescent readout probes from the plurality of the encoding probe antisense barcode transcript hybridized complexes. In embodiments, the separating includes yvashing the plurality of free antisense adapter probes and the plurality of free fluorescent readout probes from the hydrogel. In embodiments, the separating includes washing the plurality of free antisense adapter probes from the hydrogel. In embodiments, the separating includes washing the plurality of free fluorescent readout probes from the hydrogel. In embodiments, the plurality of separatedencoding probe antisense barcode transcript hybridized complexes are not removed from the hydrogel.

[0162] In embodiments, step (i) further includes contacting the plurality of separated encoding probe antisense barcode transcript hybridized complexes with a second plurality7of different antisense adapter probes and allowing each of the different antisense adapter probes in the second plurality7of different antisense adapter probes to hybridize to a different antisense encoding probe bound to the antisense barcode transcript thereby forming a second plurality of adapter probe antisense barcode transcript hybridized complexes. In embodiments, step (i) includes contacting the plurality7of separated encoding probe antisense barcode transcript hybridized complexes yvith a second plurality of different antisense adapter probes. In embodiments, step (i) includes allowing each of the different antisense adapter probes in the second plurality of different antisense adapter probes to hybridize to a different antisense encoding probe bound to the antisense barcode transcript thereby forming a second plurality7of adapter probe antisense barcode transcript hybridized complexes. In embodiments, the second plurality of different antisense probes is different from the first plurality of different antisense adapter probes.

[0163] In embodiments, step (j) further includes contacting the second plurality7of adapter probe antisense barcode transcript hybridized complexes yvith the plurality7of different fluorescent readout probes and allowing each of the different fluorescent readout probes to hybridize to a different antisense adapter probe bound to the antisense barcode transcript thereby forming a second plurality of fluorescent readout probe antisense barcode transcript hybridized complexes. In embodiments, step (j) includes contacting the second plurality of adapter probe antisense barcode transcript hybridized complexes with the plurality7of different fluorescent readout probes. In embodiments, step (j) includes allowing each of the different fluorescent readout probes to hybridize to a different antisense adapter probe bound to the antisense barcode transcript thereby forming a second plurality of fluorescent readout probe antisense barcode transcript hybridized complexes.

[0164] In embodiments, step (k) includes detecting the second plurality of different fluorescent readout probes hybridized to the antisense barcode transcript, thereby detecting the antisense barcode transcript.

[0165] In embodiments, step (g) includes denaturing the hybridized antisense adapter probes and the hybridized fluorescent readout probes from the plurality of encoding probe antisense barcode transcript hybridized complexes thereby producing the plurality of free antisense adapter probes and the plurality of free fluorescent readout probes; step (i) further includes contacting the plurality of plurality of encoding probe antisense barcode transcript hybridized complexes with a second plurality of different antisense adapter probes and allowing each of the different antisense adapter probes in the second plurality of different antisense adapter probes to hybridize to a different antisense encoding probe bound to the antisense barcode transcript thereby forming a second plurality of adapter probe antisense barcode transcript hybridized complexes; step (j) further includes contacting the second plurality of adapter probe antisense barcode transcript hybridized complexes with the plurality of different fluorescent readout probes and allowing each of the different fluorescent readout probes to hybridize to a different antisense adapter probe bound to the antisense barcode transcript thereby forming a second plurality7of fluorescent readout probe antisense barcode transcript hybridized complexes; and step (k) includes detecting the second plurality of different fluorescent readout probes hybridized to the antisense barcode transcript, thereby detecting the antisense barcode transcript.

[0166] In embodiments, the method includes repeating steps (g)-(k). In embodiments, each repeated step (i) includes contacting the plurality of encoding probe antisense barcode transcript hybridized complexes with a corresponding plurality of different antisense adapter probes, wherein the plurality of different antisense adapter probes used in each repeated step is different, and allowing each of the plurality of different antisense adapter probes in each repeated step to hybridize to a different antisense encoding probe bound to the antisense barcode transcript thereby forming a corresponding plurality of adapter probe antisense barcode transcript hybridized complexes. In embodiments, each repeated step (i) includes contacting the plurality of encoding probe antisense barcode transcript hybridized complexes with a corresponding plurality of different antisense adapter probes. In embodiments, the plurality of different antisense adapter probes used in each repeated step is different. In embodiments, the method includes allowing each of the plurality of different antisense adapter probes in each repeated step to hybridize to a different antisense encoding probe bound to the antisense barcode transcript thereby forming a corresponding plurality7of adapter probe antisense barcode transcript hybridized complexes.

[0167] In embodiments, each repeated step (j) includes contacting the corresponding plurality of adapter probe RNA transcript hybridized complexes with the plurality of different fluorescent readout probes and allowing each of the different fluorescent readout probes to hybridize to a different adapter probe bound to the antisense barcode transcript thereby forming a corresponding plurality of fluorescent readout probe antisense barcode transcript hybridized complexes.

[0168] In embodiments, the barcode sequence is 360 nucleotides in length. In embodiments, the barcode sequence includes 9 different barcode subsequences. In embodiments, each different barcode subsequence is 40 nucleotides in length. In embodiments, the 9 different barcode subsequences are orthogonal to each other. In embodiments, the 9 different barcode subsequences do not include homology to the human genome or the mouse genome. In embodiments, the 9 different barcode subsequences do not include homology to the human genome. In embodiments, the 9 different barcode subsequences do not include homology' to the mouse genome.

[0169] In embodiments, step (g) further includes denaturing the hybridized antisense adapter probes and the hybridized fluorescent readout probes from the plurality of encoding probe antisense barcode transcript hybridized complexes thereby producing the plurality of free antisense adapter probes and the plurality7of free fluorescent readout probes; step (i) further includes contacting the plurality of plurality of encoding probe antisense barcode transcript hybridized complexes with a second plurality of different antisense adapter probes and allowing each of the different antisense adapter probes in the second plurality of different antisense adapter probes to hybridize to a different antisense encoding probe bound to the antisense barcode transcript thereby forming a second plurality7of adapter probe antisense barcode transcript hybridized complexes; step (j) further includes contacting the second plurality of adapter probe antisense barcode transcnpt hybridized complexes with the plurality of different fluorescent readout probes and allowing each of the different fluorescent readout probes to hybridize to a different antisense adapter probe bound to the antisense barcode transcript thereby forming a second plurality of fluorescent readout probe antisense barcode transcript hybridized complexes; step (k) further includes detecting the second plurality of different fluorescentreadout probes hybridized to the antisense barcode transcript, thereby detecting the antisense barcode transcript.

[0170] In embodiments, the method includes contacting the plurality of encoding probe RNA transcript hybridized complexes and / or the plurality of encoding probe antisense transcript hybridized complexes with a corresponding plurality of different adapter probes and / or a plurality of different antisense adapter probes; and allowing each adapter probe within the plurality of different adapter probes to hybridize to a encoding probe RNA transcript complex within the plurality of encoding probe RNA transcript hybridized complexes, thereby forming a plurality of adapter probe RNA transcript hybridized complexes and / or allowing each antisense adapter probe within the plurality of different antisense adapter probes to hybridize to an encoding probe antisense barcode transcript complex within the plurality of encoding probe antisense barcode transcript complexes, thereby forming a plurality of adapter probe antisense barcode transcript complexes.

[0171] In embodiments, the method includes contacting the plurality of adapter probe RNA transcript hybridized complexes and / or the plurality of adapter probe antisense transcript hybridized complexes with a corresponding plurality of different fluorescent readout probes; and allowing each fluorescent readout probe within the plurality of different fluorescent readout probes to hybridize to an adapter probe RNA transcript hybridized complex within the plurality of adapter probe RNA transcript hybridized complexes, thereby forming a plurality of fluorescent readout probe RNA transcript hybridized complexes and / or allowing each fluorescent readout probe within the plurality of different fluorescent readout probes to hybridize to an adapter probe antisense barcode transcript complex within the plurality of adapter probe antisense barcode transcript complexes, thereby forming a plurality of fluorescent readout probe antisense barcode transcript complexes.

[0172] In embodiments, the detecting includes the use of a fluorescent microscopemicrofluidics system. In embodiments, the detecting includes contacting the plurality of fluorescent readout probe RNA transcript hybridized complexes with an imaging buffer. In embodiments, the detecting includes contacting the plurality of fluorescent readout probe antisense barcode transcript hybridized complexes with an imaging buffer. In embodiments, the detecting includes contacting the plurality of fluorescent readout probe RNA transcripthybridized complexes and the plurality of fluorescent readout probe antisense barcode transcript hybridized complexes with an imaging buffer. In embodiments, the detecting includes 16 repeated iterations of detecting the corresponding plurality of fluorescent readout probe RNA transcript complexes and / or the corresponding plurality' of fluorescent readout probe antisense barcode transcript complexes.

[0173] In embodiments, the detecting of the plurality of different fluorescent readout probes hybridized to the barcoded RNA transcript includes exciting each chemical fluorophore within each different fluorescent readout probe with a specific excitation light wavelength. In embodiments, each excited chemical fluorophore emits light at an emission light wavelength. In embodiments, the detecting of the plurality of different fluorescent readout probes hybridized to the barcoded RNA transcript includes detecting the light emitted by each chemical fluorophore at the emission light wavelength.

[0174] In embodiments, the detecting of the plurality of different fluorescent readout probes hybridized to the antisense barcode transcript includes exciting each chemical fluorophore within each different fluorescent readout probe with a specific excitation light wavelength. In embodiments, each excited chemical fluorophore emits light at an emission light wavelength. In embodiments, the detecting of the plurality of different fluorescent readout probes hybridized to the antisense barcode transcript includes detecting the light emitted by each chemical fluorophore at the emission light wavelength. Imaging methods for detecting fluorescent readout probes are well known in the art. For example. Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH) is a well-known imaging method known in the art. See, e.g., Chen et al., Science, 348, 6090 (2015); Moffitt et al., PNAS, 113, 11046 (2016); and / or Moffitt et al., PNAS, 113, 14456 (2016), each of which is hereby incorporated in its entirety and for all purposes.

[0175] In embodiments, the detecting of the plurality of different fluorescent readout probes hybridized to the barcoded RNA transcript further includes quantifying the expression of the barcoded RNA transcript. In embodiments, the detecting of the plurality' of different fluorescent readout probes hybridized to the antisense barcode transcript further includes quantify ing the expression of the antisense barcode transcript. In embodiments, the quantified expression of thebarcoded RNA transcript is normalized to the quantified expression of the antisense barcode transcript.

[0176] In embodiments, the incubation of probes and hybridized complexes provided herein including embodiments thereof occurs at a temperature between about 4 °C and about 37 °C. In embodiments, the incubation of probes and hybridized complexes provided herein including embodiments thereof occurs at a temperature of about 37 °C. In embodiments, the incubation of probes and hybridized complexes provided herein including embodiments thereof occurs for between about 30 minutes and about 18 hours. In embodiments, the incubation of probes and hybridized complexes provided herein including embodiments thereof occurs for about 30 minutes. In embodiments, the incubation of probes and hybridized complexes provided herein including embodiments thereof occurs for about 1.5 hours. In embodiments, the incubation of probes and hybridized complexes provided herein including embodiments thereof occurs for about 4 hours. In embodiments, the incubation of probes and hybridized complexes provided herein including embodiments thereof occurs for about 8 hours. In embodiments, the incubation of probes and hybridized complexes provided herein including embodiments thereof occurs overnight.

[0177] In another aspect is provided a method of quantifying transcriptional activity in a cell, the method including: (i) transfecting the cell with a reporter construct; (ii) allowing the cell to express the reporter construct; (iii) contacting the cell with a probe; (iv) quantifying expression of the reporter construct, thereby quantifying transcriptional activity in the cell.

[0178] In another aspect is provided a method of quantifying cis-regulatory element activity in a cell, the method including: (i) transfecting the cell with a reporter construct(ii) allowing the cell to express the reporter construct; (iii) contacting the cell with a probe; (iv) quantifying expression of the reporter construct, thereby quantifying cis-regulatory element activity in the cell.

[0179] In embodiments, the reporter construct includes: (i) a nucleic acid encoding a cis- regulatory element (CRE); and (2) a barcode.

[0180] In embodiments, the barcode includes a barcode nucleotide sequence.

[0181] In embodiments, the barcode nucleotide sequence is between about 40 to about 360 nucleotides in length.

[0182] In embodiments, the reporter construct includes a plasmid.

[0183] In embodiments, the probe is complementary to the barcode.

[0184] In embodiments, the probe is a fluorescent probe.

[0185] In embodiments, the quantifying of step (iv) includes fluorescent microscopy.

[0186] In embodiments, the quantifying of step (iv) includes whole cell segmentation.

[0187] In embodiments, the quantifying of step (iv) includes fluorescent in situ hybridization (FISH).

[0188] In embodiments, the quantifying of step (iv) is repeated one or more times.

[0189] In embodiments, the cell is transfected with one or more reporter constructs. In embodiments, the one or more reporter constructs include a nucleic acid encoding the same cis- regulatory element. In embodiments, the one or more reporter constructs include a nucleic acid encoding different cis-regulatory elements. In embodiments, the one or more reporter constructs include the same barcode. In embodiments, the one or more reporter construct include different barcodes.

[0190] In embodiments the cell is contacted with one or more probes. In embodiments, the one or more probes target the same barcode. In embodiments, the one or more probes target different barcodes.EXPRESSION VECTOR COMPOSITIONS

[0191] The compositions provided herein include nucleic acid compositions. The nucleic acid compositions provided herein including embodiments thereof include expression vector compositions. The expression vector compositions provided herein are useful for, inter alia, methods of detecting transcriptional activity of a cis regulator ' element (e.g., an enhancer). The expression vector compositions provided herein are described in detail throughout this application (including the description above and in the examples section). Thus, in an aspect isprovided an expression vector including a DNA sequence encoding a protein encoding sequence, an enhancer sequence, and a barcode sequence.

[0192] The expression vector compositions provided herein including embodiments thereof may form part of a kit. Thus, in another aspect is provided a kit including the expression vector provided herein including embodiments thereof.EXAMPLESExample 1: Summary

[0193] Current tools for functional characterization of cis elements and regulatory variants suffer from several major limitations: they often rely on in vitro models that fail to represent the diversity of cell types and conditions in the human body; the existing in vivo reporter assays using transgenic animals lack scalability needed for analysis of the millions of candidate cis regulatory elements; the existing tools provide insufficient resolution for understanding the context-dependent and cell-type specific functions. These limitations severely constrain our ability to interpret the role of non-coding DNA variants in disease, stalling progress in study of a wide spectrum of human disease.

[0194] The current invention aims to bridge the knowledge gap in analyzing the cis-regulatory code in the genome. The method, STARR-FISH, a spatially resolved, single-cell based, high- throughput reporter assay, overcomes major limitations of existing methodologies. STARR- FISH combines an ultra-high throughput assay for testing enhancer activities first introduced in STARR-seq1, with multiplexed error-robust fluorescence in situ hybridization (MERFISH) for single cell spatial transcriptomics2The key innovations of STARR-FISH are: (1) creative use of MERFISH to enable single-cell resolution, spatially resolved and multiplexed analysis of transcriptional activities of many cCREs in parallel; (2) innovative approach to associate cCREs with combinatorial molecular barcodes for MERFISH; (3) novel analytical techniques to decode and quantity activities of cCREs in situ.Example 2: Background

[0195] Deciphering the cis regulatory' code, which governs how the DNA sequences interact with sequence-specific transcription factors and chromatin binding proteins to mediate cell-type specific gene expression patterns, is a central question in molecular biology. While millions ofcandidate CREs (cCREs) have been identified in the human genome through genome-wide profiling of biochemical marks such as chromatin accessibility, DNA hypomethylation, and various histone modifications3- 4, the vast maj ori ty of them remain to be functionally characterized. The sheer number of the cCREs combined calls for development of innovative high-throughput functional characterization technologies for assessing the transcriptional activities across numerous cell states and physiological conditions.

[0196] Several high-throughput assays have been developed for functional characterization of cis-regulatory elements (CREs)5. Among them are the Massively Parallel Reporter Assay (MPRA) for high-throughput enhancer screening with unique DNA barcodes6- 7, and STARR- seq1, which simplifies this process by integrating enhancer sequences directly downstream of a promoter for genome-wide analysis. Single-cell MPRA (scMPRA)8extends this approach to the single-cell level, utilizing barcodes to track the activity of specific regulatory sequences and their impact on gene expression across various cell types. Meanwhile, the Paralleled Enhancer Single Cell Assay (PESCA)9leverages barcoded AAV vectors analyzed via single-nucleus RNA sequencing to assess the specificity of genomic regulatory elements in a highly detailed manner, across an extensive range of cell types. Each of these methods provides a unique approach to elucidate the functions of CREs, offering insights into their regulatory roles across different cellular contexts and stages of development.

[0197] On the other hand, existing methods still face significant limitations that impede a full understanding of the cCREs in the genome. Current techniques often struggle with cell-type specificity as they typically rely on in vitro cell cultures and bulk analyses, which fail to capture the diversity of cell types and physiological conditions in the human body. Moreover, the integration of these assays with the complexity of tissue environments remains challenging, as does detailing reporter gene activities within heterogeneous cell populations. The dynamic range and detection efficiency of these methods are further constrained by suboptimal mRNA capture in single-cell assays and inefficient DNA delivery to target cell populations, which can obscure subtle regulatory effects. Lastly, the scalability- and resolution of traditional techniques, such as reporter assays with transgenic animals10, are insufficient for analyzing millions of cCREs or dissecting their context-dependent and cell-type-specific functions. These constraints significantly impede our ability to thoroughly investigate cCREs and elucidate the contributionsof non-coding DNA variants to human diseases. Innovative approaches that enable more precise and comprehensive analyses of cis-regulatory elements in their native cellular and tissue contexts are desperately needed.Example 3: STARR-FISH

[0198] Our invention, STARR-FISH (FIGS. 1A-1C), is a spatially resolved, single-cell based, high-throughput reporter assay that overcomes the limitations of existing methodologies. STARR-FISH combines the high throughput strategy for testing enhancer activities first introduced in STARR-seq1, with the multiplexed error-robust fluorescence in situ hybridization (MERFISH)2for single cell spatial transcriptomics. In proof of principle experiments detailed below, we have demonstrated the superior performance of STARR-FISH in quantification of enhancer activities at single-cell resolution while preserving spatial information.

[0199] The premise of STARR-FISH is similar to that of STARR-seq1, whereby a reporter gene (for example SYFP2), the enhancer sequence and its associated barcode are transcribed from a minimal promoter in a manner dependent on a given enhancer’s transcriptional activity. A key innovation here is the design of 360-bp modular DNA barcodes that are composed of nine randomly assembled 40-mer probe sequences, which are orthogonal to each other and with no homology to both the human and mouse genomes (FIG. 1A). Plasmid libraries containing the enhancers and barcodes will be designed and constructed via a traditional molecular cloning approach, and delivered to cells or live animals via transfection or viral injection, respectively. Transcripts originating from the plasmids will be imaged via sequential or multiplexed RNA FISH using 40-mer probes targeting both the barcode sequences and SYFP2 (FIG. IB). These probes can then be stripped from the sample, facilitating multiple rounds of imaging as previously described2. Subsequently, the samples will be subject to sequential or multiplexed DNA FISH utilizing fluorescence oligo probes corresponding to the molecular barcodes. After imaging, the detected reporter mRNA spots can be decoded based on the unique combination of barcodes and assigned to the corresponding enhancers, and the plasmid copy numbers determined based on the DNA FISH results. The ratio of reporter mRNA molecules for an enhancer and the corresponding plasmid DNA copy number in a cell provides a direct measurement of cv.s-regulatory element activity in each cell. When performed in combination of MERFISH2, STARR-FISH would enable highly quantitative and accurate determination ofenhancer activities in each constituent cell type in a mixed population or a complex tissue, therefore filling a key gap in the toolsets of functional genomics.Example 4: Results

[0200] As a proof of principle, we first carried out an experiment to test the feasibility of STARR-FISH in accurately reporting enhancer activity in HCT116 colorectal cancer cells. We selected 15 cA-regulatory elements (CREs) previously reported to have enhancer activity in these cells11and five random yeast open reading frames (ORFs) to serve as negative controls. Upon construction of the plasmid library containing these 20 regulatory elements and their assigned barcodes, we transfected HCT116 cells and began sample preparation for imaging after 24 hours. We performed sequential RNA FISH over 7 consecutive rounds of imaging. Probes targeting the barcoded mRNA transcripts revealed bright and highly resolvable spots for each individual cCRE in the library, validating the barcode detection scheme (FIG. 2A, top). The resulting spots were then assigned to cell segmentation masks, providing a measurement of the transcriptional activity (number of transcripts) for each cCRE in the library (FIG. 2A, bottom).

[0201] As expected, we observed a strong correlation between the number of total transcripts per cell and nearly all individual cCREs tested in the pilot library. This correlation was noticeably stronger for more active cCREs, as determined by the number of transcripts per cell, and weaker for cCREs with fewer transcripts per cell. Moreover, the transcript counts for nearly all cCREs correlated remarkably well within each activity group, suggesting that cells were uniformly transfected with each of the unique barcoded plasmids within the library. This observation led us to determine the relative enhancer activities by linearly regressing the singlecell STARR-FISH data. Indeed, we found that enhancer activities could be precisely calculated with narrow coefficients of variation (c.v.) (FIG. 2B). These activity scores also correlated well with bulk RNA-seq of transfected HCT116 cells, confirming that the STARR-FISH measurements accurately captured the transcript abundance for each barcoded cCRE (FIG. 2C). Importantly, enhancer activities between biological replicates also showed a strong correlation, indicating that STARR-FISH is both robust and reproducible (FIG. 2D).

[0202] To determine the scalability and power afforded by STARR-FISH in measuring enhancer activity, we next asked how many cells were required for precise and accurate quantification. We sub-sampled decreasing numbers of cells to calculate the relative enhanceractivity for each of the 20 cCREs and plotted the variation in activity scores for each subsampled population (FIG. 3). We found that using as few as 100 cells was sufficient to accurately quantify relative enhancer activity with minimal variation. This indicates that STARR-FISH is capable of measuring enhancer activity with high precision, empowering the use of high-throughput libraries to assess enhancer activity in a multiplexed fashion.

[0203] To further demonstrate the scalability of STARR-FISH, we designed and constructed a library of reporter plasmids containing 300 cCREs and controls. In each plasmid, a cCRE is linked to a unique combination of combinatorial barcode as illustrated in Figure 1. We carried out STARR-FISH assays by transfecting the library of reporters to HCT116 cells, and performed multiplexed, error-robust RNA FISH. We then decoded the reporter transcripts using the combinatorial barcodes, and quantified the abundance of each species in every cell. Finally, we computed the relative enhancer activities as described above. The results confirmed that enhancer activities could be detected with the more complex library', with high reproducibility (FIGS. 4A-4B)Example 5: Key Innovations

[0204] STARR-FISH represents a groundbreaking advancement in functional genomics by integrating high-throughput capabilities with unparalleled precision and depth of analysis. This novel methodology' promises to overcome limitations of existing tools by offering single-cell resolution alongside spatially resolved data within tissue contexts. Such a leap forward will significantly enhance our ability to functionally characterize a vast array of cCREs across diverse cell types and physiological states, marking a new era in our understanding of genomic regulation.

[0205] MERFISH Integration for Spatial Functional Genomics: The integration of Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH) within the STARR- FISH framework enables the simultaneous analysis of the transcriptional activities of a large number of cCREs at single-cell resolution. This creative combination of molecular barcoding strategy7of MERFISH and the massively' parallel reporter assays in STARR-seq allows for the spatially resolved and multiplexed analysis of cCREs, making it possible to dissect their celltype specific activities within complex tissues.

[0206] Cuting-edge Reporter Design: The development of innovative reporter constructs that link cCREs with molecular barcodes optimized for MERFISH analysis is a cornerstone of the STARR-FISH approach. This enables the precise identification and quantification of cCRE activity , leveraging the specificity7and multiplexing capability of MERFISH to characterize millions of cCREs across the genome.

[0207] Novel Analytical Techniques for in situ Decoding: STARR-FISH incorporates novel analytical strategies designed to decode and quantify the activities of cCREs directly within tissues. This in situ analysis bypasses the limitations of traditional bulk or dissociative single cell omics assays by maintaining the spatial context of cells, thereby providing insights into the dynamic and cell-type-specific regulatory function of CREs in the complex tissue environments.

[0208] Unveiling the Regulatory Code in Physiological Contexts: By offering the tools to dissect the functional impact of cCREs within their native tissue environments, STARR-FISH sets the stage for uncovering the cis-regulatory code in health and disease. Understanding how these elements interact with their surroundings to drive gene expression will illuminate new pathways for therapeutic intervention and genomic engineering.

[0209] Bridging Genomic Analysis and Tissue Architecture: The possibility of combining STARR-FISH with standard MERFISH techniques allow for generation of spatially resolved CRE activity profiles along with spatial transcriptomics data, thereby bridging the gap between high-throughput genomic analysis and the complex tissue architecture that defines biological function. This synthesis opens new avenues for studying gene regulation in health and disease.

[0210] In summary, STARR-FISH represents a paradigm shift in functional genomics, leveraging the power of advanced imaging techniques to explore the regulatory genome with unprecedented detail and scale. This innovative approach unlocks new dimensions of understanding in genomic regulation, with broad implications for biology7, medicine, and biotechnology.References

[0211] 1. Arnold. C.D. et al. Genome-wide quantitative enhancer activity maps identified by STARR-seq. Science 339, 1074-1077 (2013).

[0212] 2. Chen, K.H.. Boettiger, A.N., Moffitt. J.R., Wang, S. & Zhuang, X. RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015).

[0213] 3. Consortium, E.P. et al. Expanded encyclopaedias of DNA elements in the human and mouse genomes. Nature 583, 699-710 (2020).

[0214] 4. Zhang, K. et al. A cell atlas of chromatin accessibility across 25 adult human tissues. Cell in press (2021).

[0215] 5. Shlyueva. D.. Stampfel. G. & Stark, A. Transcriptional enhancers: from properties to genome-wide predictions. Nat Rev Genet 15, 272-286 (2014).

[0216] 6. Melnikov, A. et al. Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay. Nat Biotechnol 30, 271-277 (2012).

[0217] 7. Patwardhan, R.P. et al. Massively parallel functional dissection of mammalian enhancers in vivo. Nat Biotechnol 30, 265-270 (2012).

[0218] 8. Zhao, S. et al. A single-cell massively parallel reporter assay detects cell-type- specific gene regulation. Nat Genet 55, 346-354 (2023).

[0219] 9. Hrvatin, S. et al. A scalable platform for the development of cell-type-specific viral drivers. Elife 8 (2019).

[0220] 10. Visel, A., Rubin, E.M. & Pennacchio, L.A. Genomic views of distant-acting enhancers. Nature 461. 199-205 (2009).

[0221] 11. Chen, P.B. et al. Systematic discovery and functional dissection of enhancers needed for cancer cell fitness and proliferation. Cell Rep 41, 1 11630 (2022).Example 6: Materials and Methods

[0222] Molecular Barcode design: The barcodes used for multiplexed RNA FISH to represent individual enhancer sequences were designed from a catalog of previously validated probe sequences used in DNA microarrays. These 40 nt probe sequences are screened for ideal properties conducive to multiplexed RNA FISH experiments, including their melting temperature, lack of secondary structure, and lack of sequence similarity or binding specificitywithin the human and mouse genomes. The sequences were then computationally assembled in a random fashion to generate unique 360 bp barcodes that are orthogonal to each other (See Informal Sequence Listing). The final barcode sequences are ultimately designed to be robustly and specifically targeted during our multiplexed RNA FISH experiments with minimal to no off- target hybridization events, resulting in low background during imaging.

[0223] Plasmid library construction: Plasmids containing matched enhancers (cis-regulatory elements, or CREs) and molecular barcodes were assembled through standard molecular cloning approaches (Figure 2). Briefly, oligo pools containing either the enhancer fragments or the molecular barcodes were ordered from IDT (See Informal Sequence Listing). Importantly, the CRE-barcode association was determined a priori, eliminating the need for downstream identification of matched CRE-barcode pairs. Oligos from each pool were first assembled via PCR, where fragments containing a given CRE were joined with their assigned barcode fragment resulting in double stranded CRE-barcode fragments. Full length CRE-barcode DNA fragments were subsequently assembled into the digested STARR-FISH plasmid backbone via isothermal assembly and electroporated into competent, recombination-deficient bacteria. Thousands of individual colonies containing a single, unique CRE-barcode reporter plasmid were pooled together to generate STARR-FISH reporter plasmid libraries for each experiment.

[0224] Probe synthesis: Encoding probes used in STARR-FISH assays were amplified by limited cycles of PCR from oligo pools synthesized by Twist Bioscience (See Informal Sequence Listing). Following initial amplification, we performedin vitro transcription of the DNA probes with T7 RNA polymerase. This step provides a significant secondary amplification of the probes via conversion to RNA while minimizing potential errors introduced by PCR. Single stranded DNA probes were then reverse transcribed from the RNA template using a primer that contains an acrydite modification at the 5' end. This modification facilitates anchoring of the DNA probes into an acrylamide gel post-hybridization. Following reverse transcription, RNA was removed from the DNA probes via alkaline hydrolysis and the final probes were column purified. Probe quality was assessed via a 4200 TapeStation system (Agilent) to ensure successful synthesis.

[0225] In vitro STARR-FISH sample preparation: Cells were cultured on #1.5 coverslips that were first coated with a layer of silane and poly-L-Lysine. Following a light fixation of the cells with methanol and acetic acid, we performed in situ transcription with T7 RNA polymeraseto produce mRNA transcripts that contain the antisensebarcode for 3-16 hours at 40°C. Cells were then fixed in 4% paraformaldehyde and permeabilized with 0.05% Triton X-100 followed by 80% ethanol. Samples were subsequently incubated in a pre-hybridization buffer containing 40% formamide, 2X SSC, 0.1% Tween, and RNase inhibitor prior to probe hybridization. Encoding probes (~0.6 ng / probe) targeting the barcoded enhancer-derived transcripts and T7- derived transcripts were hybridized to the sample overnight at 37°C in a buffer containing 50% formamide, 2X SSC, 0.1% Tween, 10% dextran sulfate, and RNase inhibitor. The next day, samples were washed in pre-hybridization buffer and embedded in 4% polyacrylamide to enable anchoring of the encoding probes into the gel. Following gel embedding, samples w ere postfixed with 4% paraformaldehyde in 2X SSC before incubating 24-48h at 37°C in a tissue clearing solution containing 2.5% SDS, 0.5% Triton X-100, and 1% Proteinase K in 2X SSC. Finally, samples were thoroughly washed in 2X SSC and stored at 4°C before imaging.

[0226] In vivo experiments: The STARR-FISH plasmid library of 400 cCREs was packaged as a pool into AAV-PHP.eB particles and purified via a cesium chloride gradient. Approximately 3x1012 AAV genomes were administered to 6-8 week old C57BL / 6 male mice retro-orbitally. After four weeks, animals were sacrificed and intact brains were collected and immediately frozen in OCT until sectioning.

[0227] In vivo STARR-FISH sample preparation: Mouse brains were sagitally sectioned into 18-pm thick slices on a Leica CM3050 S Cryostat and mounted to #1.5 coverslips that have been coated with a layer of silane and poly-L-Lysine. Immediately following sectioning, tissue sections were fixed with 4% paraformaldehyde in PBS and permeabilized overnight with 80% ethanol. Samples were subsequently incubated in a pre-hybridization buffer containing 40% formamide, 2X SSC, 0.1% Tween, and RNase inhibitor prior to probe hybridization. Encoding probes (-0.6 ng / probe) targeting the barcoded enhancer-derived transcripts and cell t pe marker transcripts were hybridized overnight at 37°C in a buffer containing 50% formamide. 2X SSC. 0.1% Tween, 10% dextran sulfate, and RNase inhibitor. The next day, samples were washed in pre-hybridization buffer and embedded in 4% polyacrylamide overnight to enable anchoring of the encoding probes into the gel. Following gel embedding, samples were post-fixed with 4% paraformaldehyde in 2X SSC for 10 minutes, thoroughly washed in 2X SSC and stored at 4°C before imaging.

[0228] Bulk sequencing: Briefly, RNA and DNA from transfected cells were jointly harvested to prepare sequencing libraries. Bulk RNA-seq libraries were prepared using the Illumina Tru- seq mRNA capture kit. For DNA libraries, the plasmid region containing the enhancer and its associated barcode were amplified with primers that contain Illumina adapter sequences using a limited number of PCR cycles. Both RNA and DNA libraries were sequenced on a Nextseq 2000. Paired-end sequencing reads were mapped to a custom reference genome containing the enhancer-barcode sequences.

[0229] Image acquisition: MERFISH(l) was carried out as previously described, where each round of imaging consisted of the following: 1) adapter probes were hybridized to the sample for ~1.5 hours in a buffer containing 35% formamide. 2X SSC, and 0.1% Tween; 2) the sample was washed with 30% formamide in 2X SSC and 0. 1% Tween; 3) readout probes were hybridized to the sample for -30 minutes in a buffer containing 35% formamide, 2X SSC, and 0. 1% Tween; 4) the sample was washed with 30% formamide in 2X SSC and 0.1% Tween; 5) imaging buffer was flowed onto the sample for image acquisition: 6) readout and adapter probes were stripped from the sample using a buffer containing 80% formamide and 0.8X SSC. For all STARR-FISH experiments, we captured z-stacks of each field of view (FOV) in 4 colors: 750 nm (Alexa 750), 647 nm (Cy5), and 560 nm (Cy3) to acquire MERFISH signals for mRNA transcripts, and 405 nm (DAPI) for nuclei-based image registration. Images were acquired in all color channels before moving the stage in the z plane for each FOV at a rate of 10-20 Hz. Images were acquired using custom-built microscope-microfluidics systems, built around an ASI microscope body with a Nikon CFI Plan Apo Lambda 60x oil immersion objective. All system components are controlled using custom software developed by the Zhuang lab (github.com / ZhuangLab / storm- control).

[0230] Decoding of individual RNA molecules: All raw data processing was done using custom Python scripts. Briefly, the images are first aligned across hybridization rounds using the DAPI (nuclei) channel to adjust for x-, y-, and z-drift in the position of the stage between imaging rounds. Following flat field correction, local maxima (spots) were fitted and grouped into clusters, where each cluster contains all spots or molecules from each imaging round within a 2-pixel radius of an anchor spot. Clusters are generated for every possible anchor spot, and those containing spots from at least four images were assigned a gene identity according to thebest matching binary barcode from the MERFISH codebook. Clusters were then filtered to remove any potential false positives by comparing the mean brightness, pixel size, and distance to the closest barcode of clusters assigned to blank barcodes to those assigned to genes in order to achieve an estimated misidentification rate of 5%. The exact position of each decoded RNA molecule was calculated as the median position of all pixels consisting of the molecule.

[0231] STARR-FISH activity quantification: Cellpose(2) was used to perform image segmentation to determine the cell boundaries. Nuclei were segmented with the ‘nuclei’ model on the DAPI channel, while cytoplasm boundaries for in vitro experiments were segmented with a trained model using a polyT stain. Decoded RNA molecules were assigned to cells by applying the segmentation masks to the spatial coordinates of the molecules. Segmentation masks that did not contain any decoded RNA molecules were removed before constructing the final cell-by- gene matrices. For in vitro data, transcript counts for the barcoded enhancers were subjected to a simple linear regression against either the total transcript or negative control transcript counts per cell, then normalized toplasmid copy numbers inferred from either long-read sequencing of the plasmid library used or antisense, T7-driven barcode imaging. For in vivo data, transcript counts for the barcoded enhancers were subjected to a simple linear regression against the total negative control counts per cell, then normalized to plasmid copy numbers inferred from long-read sequencing of the plasmid library used to package AAV. Scanpy(3) was used to cluster mouse brain cells based on the marker gene transcript counts. All analysis was done using custom Python scripts, and image data was displayed using Napari.References

[0232] 1. K. H. Chen, A. N. Boettiger, J. R. Mo6itt, S. Wang, X. Zhuang, RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015).

[0233] 2. C. Stringer, T. Wang, M. Michaelos, M. Pachitariu, Cellpose: a generalist algorithm for cellular segmentation. Nat Methods 18, 100-106 (2021).

[0234] 3. F. A. Wolf, P. Angerer, F. J. Theis, SCANPY: large-scale single-cell gene expression data analysis. Genome Biol 19. 15 (2018)P EMBODIMENTS

[0235] P Embodiment 1. A method of quantifying transcriptional activity in a cell, the method comprising: (i) transfecting the cell with a reporter construct; (ii) allowing the cell to express the reporter construct; (iii) contacting the cell with a probe; (iv) quantifying expression of the reporter construct, thereby quantifying transcriptional activity in the cell.

[0236] P Embodiment 2. A method of quantifying cis-regulatory element activity in a cell, the method comprising: (i) transfecting the cell with a reporter construct; (ii) allowing the cell to express the reporter construct; (iii) contacting the cell with a probe; (iv) quantifying expression of the reporter construct, thereby quantify ing cis-regulatory element activity in the cell.

[0237] P Embodiment 3. The method of P embodiment 1 or 2, wherein the reporter construct comprises: (i) a nucleic acid encoding a cis-regulatory element (CRE); and (2) a barcode.

[0238] P Embodiment 4. The method of any one of P embodiments 1-3, wherein the barcode comprises a barcode nucleotide sequence.

[0239] P Embodiment 5. The method of any one of P embodiments 1-4, wherein the barcode nucleotide sequence is between about 40 to about 360 nucleotides in length.

[0240] P Embodiment 6. The method of any one of P embodiments 1-5, wherein the reporter construct comprises a plasmid.

[0241] P Embodiment 7. The method of any one of P embodiments 3-6, wherein the probe is complementary' to the barcode.

[0242] P Embodiment 8. The method of any one of P embodiments 1-7, wherein the probe is a fluorescent probe.

[0243] P Embodiment 9. The method of any one of P embodiments 1-8, wherein the quantifying of step (iv) comprises fluorescent microscopy.

[0244] P Embodiment 10. The method of any one of P embodiments 1-9, wherein the quantifying of step (iv) comprises whole cell segmentation.

[0245] P Embodiment 11. The method of any one of P embodiments 1-10, wherein the quantifying of step (iv) comprises fluorescent in situ hybridization (FISH).

[0246] P Embodiment 12. The method of any one of P embodiments 1 -1 1 , wherein the quantifying of step (iv) is repeated one or more times.EMBODIMENTS

[0247] Embodiment 1. A method of detecting transcriptional activity of an enhancer sequence in a cell, the method comprising: (a) delivering a plasmid to a cell, wherein said plasmid comprises a DNA sequence including a protein encoding sequence, an enhancer sequence and a barcode sequence associated with said enhancer sequence and allowing said cell to transcribe said plasmid thereby producing a barcoded RNA transcript comprising said protein encoding sequence, said enhancer sequence, and said barcode sequence, wherein said barcode sequence is operably linked to said enhancer sequence; (b) fixing the cell; (c) contacting said barcoded RNA transcript with a plurality of different encoding probes and allowing each of said plurality of different encoding probes to hybridize to a different subsequence within said barcoded RNA transcript thereby forming a plurality of encoding probe RNA transcript hybridized complexes; (d) contacting said plurality of encoding probe RNA transcript hybridized complexes with a first plurality of different adapter probes and allowing each of said different adapter probe in the first plurality of different adapter probes to hybridize to a different encoding probe bound to said barcoded RNA transcript thereby forming a first plurality of adapter probe RNA transcript hybridized complexes; (e) contacting said first plurality of adapter probe RNA transcript hybridized complexes with a plurality of different fluorescent readout probes and allowing each of said different fluorescent readout probes to hybridize to a different adapter probe bound to said barcoded RNA transcript thereby forming a first plurality of fluorescent readout probe RNA transcript hybridized complexes ; (1) detecting said first plurality of different fluorescent readout probes hybridized to said barcoded RNA transcript, thereby detecting said barcoded RNA transcript and said enhancer transcriptional activity.

[0248] Embodiment 2. The method of embodiment 1, further comprising following step (1): (g) denaturing the hybridized adapter probes and the hybridized fluorescent readout probes from the plurality of encoding probe RNA transcript hybridized complexes, thereby producing aplurality of free adapter probes and a plurality of free fluorescent readout probes; (h) separating the plurality of free adapter probes and the plurality of free fluorescent readout probes thereby providing a plurality of separated encoding probe RNA transcript hybridized complexes; (i) contacting said plurality of separated encoding probe RNA transcript hybridized complexes with a second plurality of different adapter probes and allowing each of said different adapter probe in the second plurality- of different adapter probes to hybridize to a different encoding probe bound to said barcoded RNA transcript thereby forming a second plurality of adapter probe RNA transcript hybridized complexes, wherein said second plurality of different adapter probes are different from said first plurality of different adapter probes; (j) contacting said second plurality of adapter probe RNA transcript hybridized complexes with the plurality of different fluorescent readout probes and allowing each of said different fluorescent readout probes to hybridize to a different adapter probe bound to said barcoded RNA transcript thereby forming a second plurality- of fluorescent readout probe RNA transcript hybridized complexes; (k) detecting said second plurality- of different fluorescent readout probes hybridized to said barcoded RNA transcript, thereby detecting said barcoded RNA transcript and said enhancer transcriptional activity^.

[0249] Embodiment 3. The method of embodiment 2, further comprising repeating steps (g)-(k), wherein each repeated step (i) comprises contacting said plurality of encoding probe RNA transcript hybridized complexes with a corresponding plurality of different adapter probes, wherein the corresponding plurality of different probes used in each repeated steps is different, and allowing each of said corresponding plurality of different adapter probes in each repeated step to hybridize to a different encoding probe bound to said barcoded RNA transcript thereby forming a corresponding plurality of adapter probe RNA transcript hybridized complexes.

[0250] Embodiment 4. The method of any one of embodiments 1-3, wherein each encoding probe in the plurality of encoding probes comprises a barcode-binding sequence and an adapter-binding sequence, wherein the barcode-binding sequence is capable of hybridizing to a complementary barcode subsequence within the barcoded RNA transcript.

[0251] Embodiment 5. The method of any one of embodiments 1-4, wherein the cell that is fixed forms part of a tissue comprising the cell.

[0252] Embodiment 6. The method of any one of embodiments 1-5, wherein following step (c) and prior to step (d), the method further comprises transferring the pl urality of encoding probe RNA transcript hybridized complexes to a hydrogel.

[0253] Embodiment 7. The method of any one of embodiments 1-6, the DNA sequence comprises from 5' to 3' the protein encoding sequence, the enhancer sequence, and the barcode sequence.

[0254] Embodiment 8. The method of any one of embodiments 1-7, wherein the DNA sequence further comprises a T7 polymerase promoter sequence 3' of the barcode sequence.

[0255] Embodiment 9. The method of embodiment 8, wherein following step (b) and prior to step (c) the method further comprises contacting the DNA sequence with a T7 polymerase, thereby transcribing an antisense barcode transcript.

[0256] Embodiment 10. The method of embodiment 9, wherein step (c) further comprises contacting said antisense barcode transcript with a plurality of different antisense encoding probes and allowing each of said different antisense encoding probes to hybridize to a different subsequence within said antisense barcode transcript thereby forming a plurality of encoding probe antisense barcode transcript hybridized complexes; wherein step (d) further comprises contacting said plurality of encoding probe antisense barcode transcript hybridized complexes with a first plurality of different antisense adapter probes and allowing each of said different antisense adapter probes to hybridize to a different antisense encoding probe bound to said antisense barcode transcript thereby forming a first plurality of adapter probe antisense barcode transcript hybridized complexes; wherein step (e) further comprises contacting said first plurality of adapter probe antisense barcode transcript hybridized complexes with a first plurality of different fluorescent readout probes and allowing each of said different fluorescent readout probes to hybridize to a different adapter probe bound to said antisense barcode transcript thereby forming a first plurality of fluorescent readout probe antisense barcode transcript hybridized complexes; and wherein step (f) further comprises detecting said first plurality of different fluorescent readout probes hybridized to said antisense barcode transcript thereby detecting said antisense barcode transcript.

[0257] Embodiment 11. The method of embodiment 10, wherein step (g) further comprises denaturing the hybridized antisense adapter probes and the hybridized fluorescent readout probes from the plurality of encoding probe antisense barcode transcript hybridized complexes thereby producing the plurality of free antisense adapter probes and the plurality of free fluorescent readout probes; wherein step (i) further comprises contacting said plurality of plurality of encoding probe antisense barcode transcnpt hybridized complexes with a second plurality of different antisense adapter probes and allowing each of said different antisense adapter probes in the second plurality- of different antisense adapter probes to hybridize to a different antisense encoding probe bound to said antisense barcode transcript thereby forming a second plurality of adapter probe antisense barcode transcript hybridized complexes; wherein step (j) further comprises contacting said second plurality of adapter probe antisense barcode transcript hybridized complexes with the plurality of different fluorescent readout probes and allowing each of said different fluorescent readout probes to hybridize to a different antisense adapter probe bound to said antisense barcode transcript thereby forming a second plurality of fluorescent readout probe antisense barcode transcript hybridized complexes; and wherein step (k) further comprises detecting said second plurality of different fluorescent readout probes hybridized to said antisense barcode transcript, thereby detecting said antisense barcode transcript.

[0258] Embodiment 12. The method of embodiment 11, further comprising repeating steps (g)-(k). wherein each repeated step (i) comprises contacting said plurality of encoding probe antisense barcode transcript hybridized complexes with a corresponding plurality of different antisense adapter probes, wherein the plurality of different antisense adapter probes used in each repeated step is different, and allowing each of said plurality of different antisense adapter probes in each repeated step to hybridize to a different antisense encoding probe bound to said antisense barcode transcript thereby forming a corresponding plurality of adapter probe antisense barcode transcript hybridized complexes.

[0259] Embodiment 13. The method of any one of embodiments 1-12, wherein the barcode sequence is 360 nucleotides in length.

[0260] Embodiment 14. An expression vector comprising a DNA sequence comprising a protein encoding sequence, an enhancer sequence, and a barcode sequence.

[0261] Embodiment 15. A kit comprising the expression vector of embodiment 14.INFORMAL SEQUENCE LISTINGIll

Claims

WHAT IS CLAIMED IS:

1. A method of detecting transcriptional activity of an enhancer sequence in a cell, the method comprising:(a) delivering a plasmid to a cell, wherein said plasmid comprises a DNA sequence including a protein encoding sequence, an enhancer sequence and a barcode sequence associated with said enhancer sequence and allowing said cell to transcribe said plasmid thereby producing a barcoded RNA transcript comprising said protein encoding sequence, said enhancer sequence, and said barcode sequence, wherein said barcode sequence is operably linked to said enhancer sequence;(b) fixing the cell;(c) contacting said barcoded RNA transcript with a pl ural i ty of different encoding probes and allowing each of said plurality of different encoding probes to hybridize to a different subsequence within said barcoded RNA transcript thereby forming a plurality of encoding probe RNA transcript hybridized complexes;(d) contacting said plurality of encoding probe RNA transcript hybridized complexes with a first plurality of different adapter probes and allowing each of said different adapter probe in the first plurality of different adapter probes to hybridize to a different encoding probe bound to said barcoded RNA transcript thereby forming a first plurality of adapter probe RNA transcript hybridized complexes;(e) contacting said first plurality of adapter probe RNA transcript hybridized complexes with a plurality of different fluorescent readout probes and allowing each of said different fluorescent readout probes to hybridize to a different adapter probe bound to said barcoded RNA transcript thereby forming a first plurality of fluorescent readout probe RNA transcript hybridized complexes ;(1) detecting said first plurality' of different fluorescent readout probes hybridized to said barcoded RNA transcript, thereby detecting said barcoded RNA transcript and said enhancer transcriptional activity.

2. The method of claim 1, further comprising following step (1):(g) denaturing the hybridized adapter probes and the hybridized fluorescent readout probes from the plurality of encoding probe RNA transcript hybridized complexes, thereby producing a plurality’ of free adapter probes and a plurality of free fluorescent readout probes;(h) separating the plurality of free adapter probes and the plurality of free fluorescent readout probes thereby providing a plurality of separated encoding probe RNA transcript hybridized complexes;(i) contacting said plurality of separated encoding probe RNA transcript hybridized complexes with a second plurality of different adapter probes and allowing each of said different adapter probe in the second plurality of different adapter probes to hybridize to a different encoding probe bound to said barcoded RNA transcript thereby forming a second plurality of adapter probe RNA transcript hybridized complexes, wherein said second plurality of different adapter probes are different from said first plurality of different adapter probes;(j) contacting said second plurality of adapter probe RNA transcript hybridized complexes with the plurality of different fluorescent readout probes and allowing each of said different fluorescent readout probes to hybridize to a different adapter probe bound to said barcoded RNA transcript thereby forming a second plurality of fluorescent readout probe RNA transcript hybridized complexes;(k) detecting said second plurality of different fluorescent readout probes hybridized to said barcoded RNA transcript, thereby detecting said barcoded RNA transcript and said enhancer transcriptional activity.

3. The method of claim 2, further comprising repeating steps (g)-(k), wherein each repeated step (i) comprises contacting said plurality of encoding probe RNA transcript hybridized complexes with a corresponding plurality of different adapter probes, wherein the corresponding plurality of different probes used in each repeated steps is different, and allowing each of said corresponding plurality of different adapter probes in each repeated step to hybridize to a different encoding probe bound to said barcoded RNA transcript thereby forming a corresponding plurality of adapter probe RNA transcript hybridized complexes.

4. The method of claim 1, wherein each encoding probe in the plurality of encoding probes comprises a barcode-binding sequence and an adapter-binding sequence, wherein the barcode-binding sequence is capable of hybridizing to a complementary barcode subsequence within the barcoded RNA transcript.

5. The method of claim 1, wherein the cell that is fixed forms part of a tissue comprising the cell.

6. The method of claim 1, wherein following step (c) and prior to step (d), the method further comprises transferring the plurality of encoding probe RNA transcript hybridized complexes to a hydrogel.

7. The method of claim 1, the DNA sequence comprises from 5' to 3' the protein encoding sequence, the enhancer sequence, and the barcode sequence.

8. The method of claim 1, wherein the DNA sequence further comprises a T7 polymerase promoter sequence 3' of the barcode sequence.

9. The method of claim 8. wherein following step (b) and prior to step (c) the method further comprises contacting the DNA sequence with a T7 polymerase, thereby transcribing an antisense barcode transcript.

10. The method of claim 9. wherein step (c) further comprises contacting said antisense barcode transcript with a plurality of different antisense encoding probes and allowing each of said different antisense encoding probes to hybridize to a different subsequence within said antisense barcode transcript thereby forming a plurality of encoding probe antisense barcode transcript hybridized complexes; wherein step (d) further comprises contacting said plurality of encoding probe antisense barcode transcript hybridized complexes with a first plurality of different antisense adapter probes and allowing each of said different antisense adapter probes to hybridize to a different antisense encoding probe bound to said antisense barcode transcript thereby forming a first plurality of adapter probe antisense barcode transcript hybridized complexes; wherein step (e) further comprises contacting said first plurality of adapter probe antisense barcode transcript hybridized complexes with a first plurality7of different fluorescent readout probes and allowing each of said different fluorescent readout probes to hybridize to a different adapter probe bound to said antisense barcode transcript thereby forming a first plurality of fluorescent readout probe antisense barcode transcript hybridized complexes; and wherein step (f) further comprises detecting said first plurality7of different fluorescent readout probes hybridized to said antisense barcode transcript thereby detecting said antisense barcode transcript.

11. The method of claim 10, wherein step (g) further comprises denaturing the hybridized antisense adapter probes and the hybridized fluorescent readout probes from theplurality of encoding probe antisense barcode transcript hybridized complexes thereby producing the plurality of free antisense adapter probes and the plurality of free fluorescent readout probes; wherein step (i) further comprises contacting said plurality of plurality of encoding probe antisense barcode transcript hybridized complexes with a second plurality of different antisense adapter probes and allowing each of said different antisense adapter probes in the second plurality of different antisense adapter probes to hybridize to a different antisense encoding probe bound to said antisense barcode transcript thereby forming a second plurality of adapter probe antisense barcode transcript hybridized complexes; wherein step (j) further comprises contacting said second plurality' of adapter probe antisense barcode transcript hybridized complexes with the plurality of different fluorescent readout probes and allowing each of said different fluorescent readout probes to hybridize to a different antisense adapter probe bound to said antisense barcode transcript thereby forming a second plurality of fluorescent readout probe antisense barcode transcript hybridized complexes; and wherein step (k) further comprises detecting said second plurality of different fluorescent readout probes hybridized to said antisense barcode transcript, thereby detecting said antisense barcode transcript.

12. The method of claim 11, further comprising repeating steps (g)-(k), wherein each repeated step (i) comprises contacting said plurality of encoding probe antisense barcode transcript hybridized complexes with a corresponding plurality of different antisense adapter probes, wherein the plurality of different antisense adapter probes used in each repeated step is different, and allowing each of said plurality of different antisense adapter probes in each repeated step to hybridize to a different antisense encoding probe bound to said antisense barcode transcript thereby forming a corresponding plurality of adapter probe antisense barcode transcript hybridized complexes.

13. The method of claim 1, wherein the barcode sequence is 360 nucleotides in length.

14. An expression vector comprising a DNA sequence comprising a protein encoding sequence, an enhancer sequence, and a barcode sequence.1 15. A kit comprising the expression vector of claim 14.

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