WO2025072917A1 - Barcoding intracellular reverse transcription enables high- throughput analysis of rna sequences - Google Patents

Abstract

The present invention relates to the field of nucleic acid analysis. More specifically, the present invention provides compositions and methods for multiplexing cellular analyses via intracellular cDNA barcoding. In particular embodiments, the present invention can be used to simultaneously identify RNA sequences of interest in a plurality of samples. In other embodiments, the present invention provides methods for spatial analysis of RNA sequences of interest in a fixed tissue section. In further embodiments, the present invention provides methods for detecting antibodies in a plurality of samples.

Description

BARCODING INTRACELLULAR REVERSE TRANSCRIPTION ENABLES HIGH- THROUGHPUT ANALYSIS OF RNA SEQUENCES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/586.029, filed September 28, 2024, which is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT CLAUSE

[0001]This invention was made with government support under All 18633, awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0002]The present invention relates to the field of nucleic acid analysis. More specifically, the present invention provides compositions and methods for multiplexing cellular analyses via intracellular cDNA barcoding.

BACKGROUND OF THE INVENTION

[0003]T cells recognize their target antigens via diverse repertoires of T cell receptor (TCR) sequences. The characterization of adaptive immune responses thus frequently involves defining the TCR encoding sequences present in blood or tissue.¹’⁴ The TCRp chain confers much of the antigen specificity to a T cell, and its complementarity determining region 3 (CDR3), which is the product of VDJ combinatorial and junctional diversity, is often used to uniquely define T cell clonotypes. Notably, integrating TCR sequencing with functional or phenotypic measurements, such as antigen stimulation and subsequent flow cytometric analysis, can reveal key features of adaptive immune responses.⁵’⁷

[0004]Current methods for linking TCRs to cellular phenotype can be grouped into two main categories: (i) scRNA-seq and (ii) flow cytometric sorting of cells into subpopulations for bulk repertoire sequencing. scRNA-seq enables simultaneous analysis of mRNA and protein expression within individual cells (i.e., via upstream sorting or multi- omic analyses such as Cellular Indexing of Transcriptomes and Epitopes, or CITE-seq⁸). and can define the paired alpha and beta TCR sequences necessary for reconstruction of a functional receptor. However, these approaches are limited in throughput, for example ~10⁴ cells per sample using currently available single cell technologies. Furthermore, the cost to scale these experiments to millions of cells, which may be necessary for certain applications, including identification of rare antigen-specific clones, may be prohibitive. [0005]Despite having a relatively high cellular throughput versus scRNA-seq, FACS- based separation of cellular subpopulations is somewhat limited in sample throughput. Further, separately sorted samples may suffer from imperfect comparability.⁹ For example, 30 samples sorted into four subpopulations each will result in a cumbersome 120 samples that require individual downstream processing prior to bulk sequencing. As described herein, the present invention overcomes these limitations.

SUMMARY OF THE INVENTION

[0006]The present inventors developed INtraCEllular Reverse Transcription with Sorting and sequencing (INCERTS) to increase the sample throughput and sampling depth of phenoh pe-coupled TCR repertoires. Upstream sample multiplexing is accomplished via intracellular reverse transcnption (RT) of TCR mRNA molecules using DNA barcoded primers. In this way, numerous barcoded samples can be pooled together upstream of sorting into phenotypic subpopulations for downstream repertoire sequencing. TCRp sequences are mapped back to samples of origin via their sample-specific barcodes. In the above example of 30 samples and four subpopulations of interest, the pooled barcoded samples can be sorted into just 4 subpopulations (rather than 120) for bulk repertoire sequencing. Thus, INCERTS greatly simplifies the w orkflow of otherwise overly complex experimental designs.

[0007]As further described herein, the present inventors demonstrate the utility of INCERTS for identifying potentially rare cancer-specific TCRs using PBMCs from a set of patients receiving a novel mutant KRAS peptide vaccine. 119 candidate mutant KRAS- reactive TCRs were found. The present inventors further show⁷ how⁷ integration of INCERTS with complementary patient-matched scRNA-seq data can be used to recover full-length alpha and beta chain sequences for 65 TCRs (including five of 119 candidate KRAS-reactive TCRs). Notably, INCERTs can be extended beyond T cells and TCRs to facilitate experiments requiring the analysis of millions of cells from large numbers of samples.

[0008]Accordingly, in one the present invention can be used to simultaneously identify RNA sequences of interest in a plurality⁷ of samples. In another aspect, the present invention provides methods for spatial analysis of RNA sequences of interest in a fixed tissue section. In a further aspect, the present invention provides high throughput methods for detecting antibodies in a plurality⁷ of samples.

[0009]Thus, in one aspect, the present invention provides compositions and methods useful for identifying RNA sequences of interest. In particular embodiments, a high throughput method for simultaneously identifying RNA sequences of interest in a plurality⁷ of cell samples comprises the steps of (a) fixing the cells present in the cell samples; (b) permeabilizing the cells; (c) contacting the permeabilized cells with a plurality of reverse transcription (RT) primers specific for the RNA sequences of interest under conditions that facilitate binding of RT primers to corresponding messenger RNA (rnRNA) present in the cells, wherein each RT primer comprises a sample-specific barcode; (d) reverse transcribing the rnRNA to form complementary’ deoxyribonucleic acid (cDNA) from the mRNA; (e) mixing the cell samples and sorting into cell subpopulations of interest; (f) extracting cDNA from at least one of the sorted cell subpopulations; and (g) sequencing the cDNA, wherein each sequenced cDNA comprises the RNA sequences of interest and sample-specific barcodes from step (c).

[0010]In some embodiments, prior to step (a), after any of steps (a)-(d) or as part of step (e), the method further comprises staining the cells with labeled antibodies that specifically bind cellular components including, but not limited, to cell surface markers, phenoty pic protein markers, transcription factors, chemokines or cytokines on the cell surface or intracellular, cell cycle markers, proliferation markers, pathogen derived markers, post translational modification markers, RNA probes, and the like.

[0011 ]In certain embodiments of the present invention, the sorting of step (e) comprises fluorescence-activated cell sorting (FACS) or magnetic separation. In alternative embodiments, step (d) is performed after step (e).

[0012]In a specific embodiment, after step (f), the method further comprises amplifying the cDNA using primers comprising a cell subpopulation-specific barcode, wherein each sequenced cDNA of step (g) further comprises cell subpopulation-specific barcodes. In particular embodiments, the method further comprises the step of deconvoluting the identified RNA sequences of interest back to cell subpopulation and the sample using the sample-specific barcodes and cell subpopulation-specific barcodes. In further embodiments, the method further comprises the step of using complementary patient-matched single-cell RNA-sequencing data to recover full-length sequences for the RNA sequences of interest.

[0013]The cell samples can comprise peripheral blood mononuclear cells (PBMCs). In a specific embodiment, steps (a) and (b) are performed using a single agent. In a more specific embodiment, the single agent is methanol.

[0014]In some embodiments, after step (d), the method further comprises crosslinking the cells to facilitate intracellular retention of cDNA during cell sorting. The crosslinking can be performed using dithiobis(succinimidyl propionate) (DSP). [0015]In particular embodiments, prior to step (a), after any of steps (a)-(d) or as part of step (e), the method further comprises staining the cells with a ligand for a cell surface receptor. The ligand can comprise antigen for B cells or MHC-peptide multimers for T cells.

[0016]In some embodiments, the RNA sequences of interest comprise B cell receptor (BCR) sequences. In other embodiments, the RNA sequences of interest comprise T cell receptor (TCR) sequences. In more specific embodiments, the cell samples are obtained from patients who have patients have received a candidate peptide vaccine and the method is used to identify reactive TCRs.

[0017]In another aspect, the present invention provides compositions and methods useful spatial analysis of RNA sequences of interest. In particular embodiments, a method for spatial analysis of RNA sequences of interest in a fixed tissue section comprises the steps of (a) permeabilizing the cells present in the fixed tissue section; (b) contacting the permeabilized cells with a plurality of RT primers specific for the RNA sequences of interest under conditions that facilitate binding of RT primers to corresponding mRNA present in the cells, wherein the plurality of RT primers comprises a plurality of barcodes; (c) reverse transcribing the mRNA to form cDNA; (d) determining the barcodes in situ, wherein the barcodes represent a spatial location within the cell and tissue section; (e) sequencing ex situ the cDNA comprising the RNA sequences of interest and spatial barcodes; and (f) spatially mapping the RNA sequences of interest determined in step (e) using the spatial barcodes determined in step (d). In certain embodiments, steps (c) and (d) are reversed.

[0018]In one embodiment, determining step (d) comprises performing in situ sequencing. In another embodiment, determining step (d) comprises probe-hybridization analysis. In a specific embodiment, the probe-hybridization analysis comprises ligation in situ hybridization Lock’n’Roll. In another specific embodiment, the probe hybridization analysis comprises the use of branched chain hybridization. In an alternative embodiment, the probe hybridization analysis comprises the use of hybridization chain reaction.

[0019]In a further embodiment, determining step (d) comprises imaging the spatial barcodes. In yet another embodiment, determining step (d) comprises mass spectrometry imaging analysis.

[0020]In particular embodiments, the RNA sequences of interest comprise TCR sequences, BCR sequences, cancer-associated mutations in RNA, or sequences associated with overexpressed or knocked down genes.

[0021]In a further aspect, the present invention provides methods for detecting antibodies in samples. In particular embodiments, a method for detecting antibodies in a plurality of samples comprises the steps of (a) contacting at least one sample comprising the antibodies with a plurality of cells expressing a plurality of antigens; (b) fixing the cells; (c) permeabilizing the cells; (d) contacting the permeabilized cells with a plurality of RT primers under conditions that facilitate binding of RT primers to antigen-encoding mRNA present in the cells, wherein each RT primer comprises a sample-specific barcode; (e) reverse transcribing the mRNA to form cDNA; (f) mixing the cell samples and sorting into cell subpopulations of interest based on the presence of antibodies in the samples; (g) extracting cDNA from at least one of the sorted cell subpopulations; and (h)sequencing the cDNA, wherein each sequenced cDNA comprises the antigen-encoding mRNA, sample-specific barcodes from step (d) to determine the antigen targets recognized by the antibodies in the samples.

[0022]In some embodiments, step (a) is performed after any of steps (b)-(e). In certain embodiments, the sorting of step (f) comprises FACS or magnetic separation. In other embodiments, the method further comprises the step of deconvoluting the identified antibody targets via the sample-specific barcodes. In some embodiments, the sample comprising antibodies comprises serum, plasma, saliva, or cerebrospinal fluid obtained from a patient.

[0023]In a specific embodiment, steps (b) and (c) are performed using a single agent. In a more specific embodiment, the single agent is methanol. Prior to step (I), the method can further comprise cross-linking the cells to facilitate intracellular retention of cDNA during cell sorting. In a specific embodiment, the cross-linking step is performed using dithiobis(succinimidyl propionate) (DSP).

[0024]In particular embodiments, the plurality of cells overexpresses a library of antigens. In other embodiments, the subpopulations of step (I) are defined by the presence or absence of specific antibody isotypes. In some embodiments, the antibodies in the sample comprise viral antibodies and the plurality of cells express a plurality of viral antigens. The plurality of cells can be any derive from any organism including, human, animal, bacterial or yeast.

[0025]In a specific embodiment, after step (g), the method further comprises amplilying the cDNA. In a more specific embodiment, the amplifying step utilizes primers comprising cell subpopulation-specific barcodes. In particular embodiments, the method further comprises the step of deconvoluting the identified antibody targets via the samplespecific barcodes and the cell subpopulation-specific barcodes. In alternative embodiments, cell subpopulation-specific barcodes are added any time after or as part of step (e) but before step (h). [0026]In alternative embodiments, RT primers are used target an antigen-specific barcode that is or is not encoded on the same mRNA as the antigen. Thus, in such embodiments, a method for detecting antibodies in a plurality of samples comprises the steps of (a) contacting at least one sample comprising the antibodies with a plurality of cells expressing a plurality of antigens; (b) fixing the cells; (c) permeabilizing the cells; (d) contacting the permeabilized cells with a plurality of RT primers under conditions that facilitate binding of RT primers to antigen-specific barcodes present in the cells, wherein each RT primer comprises a sample-specific barcode; (e) reverse transcribing the mRNA to form cDNA; (f) mixing the cell samples and sorting into cell subpopulations of interest based on the presence of antibodies in the samples; (g) extracting cDNA from at least one of the sorted cell subpopulations; and (h) sequencing the cDNA, wherein each sequenced cDNA comprises the antigen-encoding mRNA, sample-specific barcodes from step (d) to determine the antigen targets recognized by the antibodies in the samples.

[0027]In some embodiments, step (a) is performed after any of steps (b)-(e). In certain embodiments, the sorting of step (I) comprises FACS or magnetic separation. In other embodiments, the method further comprises the step of deconvoluting the identified antibody targets via the sample-specific barcodes and antigen-specific barcodes. In some embodiments, the sample comprising antibodies comprises serum, plasma, saliva, or cerebrospinal fluid obtained from a patient.

[0028]In a specific embodiment, steps (b) and (c) are performed using a single agent. In a more specific embodiment, the single agent is methanol. Prior to step (f), the method can further comprise cross-linking the cells to facilitate intracellular retention of cDNA during cell sorting. In a specific embodiment, the cross-linking step is performed using dithiobis(succinimidyl propionate) (DSP).

[0029]In particular embodiments, the plurality of cells overexpresses a library of antigens. In other embodiments, the subpopulations of step (I) are defined by the presence or absence of specific antibody isotypes. In some embodiments, the antibodies in the sample comprise viral antibodies and the plurality of cells express a plurality of viral antigens. The plurality of cells can be any derive from any organism including, human, animal, bacterial or yeast.

[0030]In a specific embodiment, after step (g), the method further comprises amplifying the cDNA. In a more specific embodiment, the amplifying step utilizes primers comprising cell subpopulation-specific barcodes. In particular embodiments, the method further comprises the step of deconvoluting the identified antibody targets via the sample- specific barcodes antigen-specific barcodes and the cell subpopulation-specific barcodes. In certain embodiments, the antigen-specific barcodes are encoded on the same mRNA as the antigen. In alternative embodiments, cell subpopulation-specific barcodes are added any time after or as part of step (e) but before step (h).

BRIEF DESCRIPTION OF THE FIGURES

[0031]FIG. 1A-1E. INCERTS methodology: FIG. 1A) Schematic of INCERTS protocol. Samples A and B are shown in two different wells, where they are stained with two different fluorescently conjugated antibodies. Each sample contains TCRP cDNAs generated from RT primers with sample-specific barcodes. FIG. IB) Annotated example PCR amplicons from two different sorted populations. FR4 is framework region 4. FIG. 1C) Sample-demultiplexed INCERTS data from a sorted pool of two donors, Sample A and Sample B and FIG. ID) cell population-demultiplexed INCERTS data from a single donor. Each dot corresponds to a unique CDR3 amino acid sequence and values correspond to total clonal sequencing read count. FIG. IE) Jurkat cells spiked-in at different input amounts into a background of PBMCs, in duplicate. Each point corresponds to a clonal frequency; Jurkat frequencies are shown in red and top two most abundant PBMC clone frequencies are shown in blue and green. R² and p-value of linear regression of Jurkat cell clone frequencies versus spike-in inputs are in red.

[0032]FIG. 2A-2E. INCERTS identifies mutant KRAS-reactive TCRs from vaccine trial patients: FIG. 2A) Timeline of vaccine, immunotherapy, and peripheral blood draws is shown for patients enrolled in the mutant KRAS peptide vaccine trial.³⁴ Nivo, Nivolumab; Ipi, Ipilimumab. FIG. 2B) PBMCs from 4 different donors were each split into six different peptide stimulation conditions: wild-type KRAS peptide, 4 mutant KRAS vaccine peptides (G12C. G12V, G12D, and G12A). a control EBV peptide; and a no peptide mock stimulation condition. All 28 stimulated cultures underwent the INCERTS protocol, were combined into a single pool and then sorted into four cell subpopulations. CD3⁺CD4⁺ and CD3⁺ CD8+ population gating for CD69 and CD 137 activation markers is shown. FIG. 2C) Consensus motif and sequence alignment of several mutant KRAS-reactive CDR3 sequences. FIG. 2D) Average number of total productive KRAS-reactive sequences identified via INCERTS (x- axis) is plotted against the average frequency of IFNy secreting cells detected via ELISPOT (y-axis). R² and p-value for linear regression are displayed. FIG. 2E) Contingency table showing correspondence between INCERTS-assigned cell subpopulation versus scRNA-seq- assigned cell subpopulation for CDR3s detected in donor 12 by both methods. Peptidereactive sequences are in parentheses.

[0033]FIG. 3. Schematic of In Situ INCERTS protocol for proof-of-concept spatial TCR analysis.

[0034]FIG. 4A-4H. Image analysis of In Situ INCERTS proof-of-concept studies. All images shown were obtained at 60X magnification. In FIG. 4A-4D, blue spots represent each Lock’n’Roll (LnR) Jurkat binding probe and magenta visualizes blue and green spots which overlap. In images E-G, red and green spots correspond to Jurkat binding probes and magenta indicates the overlapping Jurkat spots, whereas cyan and yellow spots correspond to Ramos binding probes and orange indicates the overlapping Ramos spots. FIG. 4A) Jurkat cells with Jurkat RT primer, Superscript IV for cDNA generation, and full LnR protocol. FIG. 4B) Jurkat cells with Jurkat RT primer but no Superscript IV, and full LnR protocol. FIG. 4C) Jurkat cells with no Jurkat RT primer, but with Superscript IV and full LnR protocol. FIG. 4D) Jurkat cells with Jurkat RT primer, Superscript IV for cDNA generation, but no T4 DNA ligase during LnR protocol. FIG. 4E) Ramos cells with Ramos RT primer, Superscript IV for cDNA generation, and full LnR protocol. FIG. 4F) Jurkat cells with mixed Jurkat and Ramos RT primers, Superscript IV for cDNA generation, and full LnR protocol. FIG. 4G) Ramos cells with mixed Jurkat and Ramos RT primers, Superscript IV for cDNA generation, and full LnR protocol. FIG. 4H) Mixed Jurkat and Ramos cells with mixed Jurkat and Ramos RT primers. Superscript IV for cDNA generation, and full LnR protocol.

[0035]FIG. 5. Ligation In Situ Hybridization Lock’n’Roll LISH-LnR Probe Configuration.

[0036]FIG. 6. Ligation In Situ Hybridization Lock’n'Roll LISH-LnR Workflow. [0037]FIG 7A-7B. FIG. 7A) Schematic showing INCERTS for antibody profiling.

FIG. 7B) Annotated example PCR amplicons from two different sorted populations.

DETAILED DESCRIPTION OF THE INVENTION

[0038]It is understood that the present invention is not limited to the particular methods and components, etc., described herein, as these may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to a “protein” is a reference to one or more proteins, and includes equivalents thereof known to those skilled in the art and so forth. [0039]Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Specific methods, devices, and materials are described, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0040]All publications cited herein are hereby incorporated by reference including all journal articles, books, manuals, published patent applications, and issued patents. In addition, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided. The definitions are not meant to be limiting in nature and serve to provide a clearer understanding of certain aspects of the present invention.

[0041]Assays linking cellular phenotypes with T cell or B cell antigen receptor sequences are crucial for characterizing adaptive immune responses. Existing methodologies are limited by low sample throughput and high cost. Such approaches include single-cell RNA-sequencing (scRNA-seq) and flow cytometric sorting of cells into subpopulations for bulk repertoire sequencing, both of which are difficult to scale across many samples and millions of cells. The present inventors have developed INtraCEllular Reverse Transcription with Sorting and sequencing, INCERTS, an approach that successfully links cellular phenoty pe with antigen receptor sequence via barcoding during reverse transcription yvithin intact cells. Subsequent cell-sorting, sequencing and demultiplexing enables high-throughput characterization of phenotype-coupled antigen receptor sequences across millions of cells.

[0042]The present inventors demonstrate that INCERTS enables efficient processing of millions of cells from pooled human PBMC samples, while retaining robust association between T cell receptor (TCR) sequences and cellular phenoty pes. The present inventors used INCERTS to discover antigen-specific TCRs from cancer patients immunized with a novel mutant KRAS peptide vaccine. After ex vivo stimulation, 28 uniquely barcoded samples were pooled prior to FACS into peptide-reactive and non-reactive CD4⁺ and CD8⁺ populations. Combining complementary' patient-matched single-cell RNA-seq data enabled retrieval of full-length, paired TCR alpha and beta chain sequences for future validation of therapeutic utility.

[0043]Although the INCERTS method provides valuable clonotypic information, it lacks spatial orientation, yvhich is frequently key to understanding immune cells’ physiologic relevance. In order to obtain information about cells and their phenotypes, as yvell as their spatial orientation in relation to other cells and tissue structures, spatial omics techniques have generated massive excitement and technical innovation. Slide-TCR-seq is one recent example of an approach that was developed to obtain spatial information about T cells, their phenotypes, and TCR sequences at lOum resolution. See Liu et al.. 55(10) IMMUNITY 1940- 52. e5 (2022). Some advantages of Slide-TCR-seq include: 1) provides transcriptomic information about all cells, which allows for phenotypic characterization, in addition to TCR sequence information for T cells, at near single-cell resolution and 2) allows for paired alphabeta TCR chain information. Some disadvantages are: 1) the beads used are proprietary and expensive, 2) the actual resolution of the assay typically spans multiple cells² and 3) TCR sequences are recovered at a relatively low efficiency of 26-42%, meaning that many T cells clones are not captured. Id.

[0044]Consequently, the present inventors adapted the INCERTS methodology to tissue sections, whereby barcoded cDNA is generated in situ, rather than inside cells in suspension. Instead of FACS sorting by phenotypic markers and sequencing the TCRs, cDNA barcodes are analyzed spatially, such that TCR cDNA can be sequenced ex situ and then mapped back to the spatial location of the associated barcode or set of barcodes. In situ determination of cDNA barcodes can be either via in situ sequencing or probe-based methods. In a specific embodiment, the present inventors demonstrate how the probe-based Ligation In Situ Hybridization Lock’n’Roll (LISH-LnR) methodology can be applied to spatial barcode identification with single cell resolution. The data suggests that clonal recovery could exceed 50%, and that by using additional probes targeting the alpha chain mRNA of the TCR, paired alpha-beta chain information can be recovered. Additionally, the approach does not require the use of proprietary reagents, enabling any laboratory to perform the technique. This In Situ INCERTS protocol is easily adaptable to spatial B cell receptor (BCR, antibody) mRNA analysis. With appropriate design of barcoded reverse transcriptase primers, the method can also preserve antibody isotype-specific information.

[0045]The description of In Situ INCERTS provided above specifically relates to the spatial localization of immune receptor sequences (TCR and BCR). However, it should be clearly stated that by no means is this a limitation of the technology. Numerous other use cases could benefit from the In Situ INCERTS invention. For example, in other embodiments, barcoded RT primers that target cancer-associated mutations could be used to create a spatial map of mutations inside a tumor. In alternative embodiments, barcoded RT primers could be designed to target cellular mRNA-containing barcodes, such as those utilized within overexpression or knockdown (e.g., RNAi and CRISPR-Cas9) screening libraries. These examples are by no means exhaustive.

I. Definitions [0046] “Detect” refers to identifying the presence, absence, amount or identity of the nucleic acid (e.g., RNA) to be detected.

[0047]As used herein, the term “labeling” refers to attaching a detectable moiety to an analyte such that the presence and/or abundance of the analyte can be determined by evaluating the presence and/or abundance of the label.

[0048]By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via. for example, spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels may include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes, biotin, digoxigenin, or haptens.

[0049]The terms “determining,” “measuring,” “evaluating,” “assessing,” “identifying,” “assaying,” and “analyzing” are used interchangeably herein to refer to forms of measurement, and include determining if an element is present or not. These terms include both quantitative and/or qualitative determinations. Assessing may be relative or absolute.

[0050]By “subj ecf ’ is meant any individual or patient to which the method described herein is applied. Generally, the subj ect is human, although as will be appreciated by those in the art, the subj ect may be an animal including mammals such as rodents (including mice, rats, hamsters and guinea pigs) and primates (including monkeys, chimpanzees, orangutans and gorillas). The term “subject” is used interchangeably with “patient.”

[0051] A “biological sample,” as used herein, is generally a sample from an individual or subject. Non-limiting examples of biological samples include blood, serum, plasma, or cerebrospinal fluid. Additionally, solid tissues, for example, spinal cord or brain biopsies may be used. In specific embodiments, a sample comprises peripheral blood mononuclear cells (PBMCs) or serum. A tissue section can also be considered a biological sample.

[0052]As used herein, the term “nucleic acid molecule” or “polynucleotide” refers to a single- or double-stranded polynucleotide containing deoxyribonucleotides or ribonucleotides that are linked by 3’ -5’ phosphodiester bonds, as well as polynucleotide analogs. A nucleic acid molecule includes, but is not limited to, DNA, RNA, and cDNA. A polynucleotide analog may possess a backbone other than a standard phosphodiester linkage found in natural polynucleotides and, optionally, a modified sugar moiety or moieties other than ribose or deoxyribose. Polynucleotide analogs contain bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, where the analog backbone presents the bases in a manner to permit such hydrogen bonding in a sequence-specific fashion between the oligonucleotide analog molecule and bases in a standard poly nucleotide.

[0053]The term “oligonucleotide” as used herein denotes a single-stranded multimer of nucleotides from about 2 to 200 nucleotides, up to 500 nucleotides in length. Oligonucleotides may be synthetic or may be made enzymatically, and, in some embodiments, are 30 to 150 nucleotides in length. Oligonucleotides may contain ribonucleotide monomers (i.e., may be oligoribonucleotides) and/or deoxyribonucleotide monomers. An oligonucleotide may be 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200 nucleotides in length, for example.

[0054]The term “primer” as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary’ to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase (e.g., reverse transcriptase) and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for some applications, depending on the complexity of the target sequence, the oligonucleotide primer may contain 15-25 or more nucleotides, although it may contain fewer nucleotides.

[0055]As used herein, a primer is “specific,” for a target sequence if, when used in an amplification reaction under sufficiently stringent conditions, the primer hybridizes primarily to the target nucleic acid. Typically, a primer is specific for a target sequence if the primertarget duplex stability’ is greater than the stability’ of a duplex formed between the primer and any other sequence found in the sample. One of skill in the art will recognize that various factors, such as salt conditions as well as base composition of the primer and the location of the mismatches, will affect the specificity of the primer, and that routine experimental confirmation of the primer specificity will be needed in many cases. Hybridization conditions can be chosen under which the primer can form stable duplexes only with a target sequence. Thus, the use of target-specific primers under suitably stringent amplification conditions enables the selective amplification of those target sequences which contain the target primer binding sites.

[0056]As described herein, primers can incorporate additional features which allow for the detection or immobilization of the primer but do not alter the basic property of the primer, that of acting as a point of initiation of DNA synthesis. For example, primers may contain an additional nucleic acid sequence at the 5' end which does not hybridize to the target nucleic acid, but which facilitates cloning, detection, or sequencing of the amplified product. The region of the primer which is sufficiently complementary to the template to hybridize is referred to herein as the hybridizing region.

[0057]As used herein, the term “barcode” refers to a nucleic acid molecule of about 2 to about 100 bases (e.g., 2. 3, 4, 5. 6, 7, 8, 9. 10. 11. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,

48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,

73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92,93,94,95,96,97, 98,

99, or 100 bases) providing a unique identifier tag or origin information for a macromolecule, each macromolecule in a library of macromolecules, and the like. A barcode can be an artificial sequence or a naturally occurring sequence. The concept of the barcode is that prior to any amplification, each original target molecule is “tagged” by a unique barcode sequence. In some embodiments, the DNA sequence must be long enough to provide sufficient permutations to assign each founder molecule a unique barcode.

[0058]In certain embodiments, each barcode within a population of barcodes is different. In other embodiments, a portion of barcodes in a population of barcodes is different, e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%. 65%, 70%, 75%, 80%, 85%, 90%. 95%, 97%, or 99% of the barcodes in a population of barcodes are different.

[0059] A population of barcodes may be randomly generated or non-randomly generated. In some embodiments, a barcode contains randomized nucleotides and is incorporated into a nucleic acid. For example, a 12-base random sequence provides 4^ or 16,777,216 unique molecular identifiers for each target molecule in the sample.

[0060] In particular embodiments, barcodes can be used to computationally deconvolute multiplexed sequencing data and identify sequence derived from an individual macromolecule, sample, library, etc. [0061]Reference throughout this specification to "one embodiment'’ or “an embodiment” or “an aspect” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0062]Ranges provided herein are understood to be shorthand for all of the values w ithin the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. 14. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3,

I.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other directi on.

[0063] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

[0064]Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 109%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

II. Cells

[0065]Methods disclosed herein include, in certain embodiments, a method for in situ gene sequencing of a target nucleic acid in a cell in an intact tissue. In several embodiments, mRNA is reverse transcribed to form cDNA in a permeabilized cell. In certain embodiments, the cell is present in a population of cells. In certain other embodiments, the population of cells includes a plurality of cell types.

[0066] Cells for use in the assays of the invention can be an organism, a single cell type derived from an organism, or can be a mixture of cell types. Included are naturally occurring cells and cell populations, genetically engineered cell lines, cells derived from transgenic animals, etc. Virtually any cell type and size can be accommodated. Suitable cells include bacterial, fungal, plant and animal cells. In one embodiment of the invention, the cells are mammalian cells, e.g., complex cell populations such as naturally occurring tissues, for example blood, liver, pancreas, neural tissue, bone marrow, skin, and the like. Some tissues may be disrupted into a monodisperse suspension. Alternatively, the cells may be a cultured population, e.g., a culture derived from a complex population, a culture derived from a single cell type where the cells have differentiated into multiple lineages, or where the cells are responding differentially to stimulus, and the like.

[0067] Cell types that can find use in the subject invention include stem and progenitor cells, e.g., embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, neural crest cells, etc., endothelial cells, muscle cells, myocardial, smooth and skeletal muscle cells, mesenchymal cells, epithelial cells; hematopoietic cells, such as lymphocytes, including T- cells, such as Thl T cells, Th2 T cells, cytotoxic T cells; B cells, pre-B cells, etc.; monocytes; dendritic cells; neutrophils; and macrophages; natural killer cells; mast cells, etc.; adipocytes, cells involved with particular organs, such as thymus, endocrine glands, pancreas, brain, such as neurons, glia, astrocytes, dendrocytes, etc., and genetically modified cells thereof. Hematopoietic cells may be associated with inflammatory processes, autoimmune diseases, etc., endothelial cells, smooth muscle cells, myocardial cells, etc., may be associated with cardiovascular diseases; almost any type of cell may be associated with neoplasias, such as sarcomas, carcinomas and lymphomas; liver diseases with hepatic cells; kidney diseases with kidney cells, etc.

[0068]The cells may also be transformed or neoplastic cells of different types, e.g., carcinomas of different cell origins, lymphomas of different cell types, etc. The American Type Culture Collection (Manassas, VA) has collected and makes available over 4,000 cell lines from over 150 different species, over 950 cancer cell lines including 700 human cancer cell lines. The National Cancer Institute has compiled clinical, biochemical and molecular data from a large panel of human tumor cell lines, these are available from ATCC or the NCI (Phelps et al. (1996) Journal of Cellular Biochemistry Supplement 24:32-91). Included are different cell lines derived spontaneously, or selected for desired growth or response characteristics from an individual cell line; and may include multiple cell lines derived from a similar tumor type but from distinct patients or sites.

[0069]Cells may be non-adherent, e.g., blood cells including monocytes, T cells, B- cells; tumor cells, etc., or adherent cells, e.g., epithelial cells, endothelial cells, neural cells, etc. In order to profile adherent cells, they may be dissociated from the substrate that they are adhered to, and from other cells, in a manner that maintains their ahi 11 ty to recognize and bind to probe molecules.

[0070]Such cells can be acquired from an individual using, e.g., a draw, a lavage, a wash, surgical dissection etc., from a variety of tissues, e.g., blood, marrow, a solid tissue (e.g., a solid tumor), ascites, by a variety of techniques that are known in the art. Cells may be obtained from fixed or unfixed, fresh or frozen, whole or disaggregated samples. III. Fixation and Permeabilization

[0071] As used herein, the term “tissue section” refers to a piece of tissue that has been obtained from a subject, fixed, sectioned, and mounted on a planar surface, e.g., a microscope slide.

[0072]As used herein, the term “formalin-fixed paraffin embedded (FFPE) tissue section” refers to a piece of tissue, e.g., a biopsy that has been obtained from a subject, fixed in, for example, formaldehyde or Bouin solution, embedded in wax, cut into thin sections, and then mounted on a planar surface, e.g., a microscope slide.

[0073]As used herein, the term “resin embedded tissue section” refers to a piece of tissue, e.g., a biopsy that has been obtained from a subject, fixed, (e.g., in 3-5% glutaraldehyde in 0.1 M phosphate buffer), dehydrated, infiltrated with epoxy or methacrylate resin, cured, cut into thin sections, and then mounted on a planar surface, e.g., a microscope slide.

[0074]As used herein, the term “cryosection” refers to a piece of tissue, e.g., a biopsy that has been obtained from a subject, snap frozen, embedded in optimal cutting temperature embedding material, frozen, cut into thin sections and fixed (e.g., in methanol or paraformaldehyde) and mounted on a planar surface, e.g., a microscope slide.

[0075]As used herein, the term “fixing” or “fixation” is the process of preserving biological material (e.g., tissues, cells, organelles, molecules, etc.) from decay and/or degradation. Fixation may be accomplished using any convenient protocol. Fixation can include contacting the sample with a fixation reagent (i.e., a reagent that contains at least one fixative). Samples can be contacted by a fixation reagent for a wide range of times, which can depend on the temperature, the nature of the sample, and on the fixative(s). For example, a sample can be contacted by a fixation reagent for 24 or less hours, 18 or less hours, 12 or less hours, 8 or less hours, 6 or less hours, 4 or less hours, 2 or less hours, 60 or less minutes, 45 or less minutes, 30 or less minutes, 25 or less minutes, 20 or less minutes, 15 or less minutes, 10 or less minutes, 5 or less minutes, or 2 or less minutes. [0076] A sample can be contacted by a fixation reagent at various temperatures, depending on the protocol and the reagent used. For example, in some instances a sample can be contacted by a fixation reagent at a temperature ranging from -22°C to 55°C, where specific ranges of interest include, but are not limited to 50 to 54°C, 40 to 44°C, 35 to 39°C, 28 to 32°C, 20 to 26°C, 0 to 6°C, and -18 to -22°C. In some instances a sample can be contacted by a fixation reagent at a temperature of -20°C, 4°C, room temperature (22-25°C). 30°C, 37°C, 42°C. or 52°C.

[0077]Any convenient fixation reagent can be used. Common fixation reagents include crosslinking fixatives, precipitating fixatives, oxidizing fixatives, mercurials, and the like. Crosslinking fixatives chemically join two or more molecules by a covalent bond and a wide range of cross-linking reagents can be used. Examples of suitable cross liking fixatives include but are not limited to aldehydes (e.g., formaldehyde, also commonly referred to as “paraformaldehyde” and “formalin”; glutaraldehyde; etc.), imidoesters, NHS (N- Hydroxysuccinimide) esters, and the like. Examples of suitable precipitating fixatives include but are not limited to alcohols (e.g., methanol, ethanol, etc.), acetone, acetic acid, etc. In some embodiments, the fixative is formaldehyde (i.e., paraformaldehyde or formalin). A suitable final concentration of formaldehyde in a fixation reagent is 0. 1 to 10%, 1-8%, 1-4%, 1-2%, 3-5%, or 3.5-4.5%, including about 1.6% for 10 minutes. In some embodiments the sample is fixed in a final concentration of 4% formaldehyde (as diluted from a more concentrated stock solution, e.g., 38%, 37%, 36%, 20%, 18%, 16%, 14%, 10%, 8%, 6%. etc.). In some embodiments the sample is fixed in a final concentration of 10% formaldehyde. In some embodiments the sample is fixed in a final concentration of 1% formaldehyde. In some embodiments, the fixative is glutaraldehyde. A suitable concentration of glutaraldehyde in a fixation reagent is 0. 1 to 1%. A fixation reagent can contain more than one fixative in any combination. For example, in some embodiments the sample is contacted with a fixation reagent containing both formaldehyde and glutaraldehyde.

[0078] As used herein, the terms “permeabilization” or “permeabilize” as used herein refer to the process of rendering the cells (cell membranes etc.) of a sample permeable to experimental reagents such as nucleic acid probes, antibodies, chemical substrates, etc. Any convenient method and/or reagent for permeabilization can be used. Suitable permeabilization reagents include detergents (e.g., Saponin, Triton X-100, Tween-20, etc.), organic fixatives (e.g., acetone, methanol, ethanol, etc.), enzymes, etc. Detergents can be used at a range of concentrations. For example. 0.001%-l% detergent, 0.05%-0.5% detergent, or 0. 1 %-0.3% detergent can be used for permeabilization (e.g., 0. 1 % Saponin, 0.2% tween- 20, 0.1-0.3% triton X-100, etc.). In some embodiments, methanol on ice for at least 10 minutes is used to permeabilize.

[0079]In some embodiments, the same solution can be used as the fixation reagent and the permeabilization reagent. For example, in some embodiments, the fixation reagent contains 0. l%-10% formaldehyde and 0.001%-l% saponin. In some embodiments, the fixation reagent contains 1% formaldehyde and 0.3% saponin. In particular embodiments, the solution comprises methanol, which can act as both a fixation reagent and a permeabilization reagent.

[0080] A sample can be contacted by a permeabilization reagent for a wide range of times, which can depend on the temperature, the nature of the sample, and on the permeabilization reagent(s). For example, a sample can be contacted by a permeabilization reagent for 24 or more hours, 24 or less hours, 18 or less hours, 12 or less hours, 8 or less hours, 6 or less hours, 4 or less hours, 2 or less hours, 60 or less minutes, 45 or less minutes, 30 or less minutes, 25 or less minutes, 20 or less minutes, 15 or less minutes, 10 or less minutes, 5 or less minutes, or 2 or less minutes. A sample can be contacted by a permeabilization reagent at various temperatures, depending on the protocol and the reagent used. For example, in some instances a sample can be contacted by a permeabilization reagent at a temperature ranging from -82°C to 55°C, where specific ranges of interest include, but are not limited to: 50 to 54°C. 40 to 44°C, 35 to 39°C, 28 to 32°C, 20 to 26°C, 0 to 6°C. -18 to -22 °C, and -78 to -82°C. In some instances a sample can be contacted by a permeabilization reagent at a temperature of -80°C, -20°C, 4°C, room temperature (22-25°C), 30°C, 37°C, 42°C, or 52°C.

[008 l]In some embodiments, a sample is contacted with an enzy matic permeabilization reagent. Enzymatic permeabilization reagents that permeabilize a sample by partially degrading extracellular matrix or surface proteins that hinder the permeation of the sample by assay reagents. Contact with an enzymatic permeabilization reagent can take place at any point after fixation and prior to target detection. In some instances the enzymatic permeabilization reagent is proteinase K, a commercially available enzyme.

[0082]In such cases, the sample is contacted with proteinase K prior to contact with a post fixation reagent. Proteinase K treatment (i.e., contact by proteinase K; also commonly referred to as “proteinase K digestion”) can be performed over a range of times at a range of temperatures, over a range of enzyme concentrations that are empirically determined for each cell type or tissue type under investigation. For example, a sample can be contacted by proteinase K for 30 or less minutes, 25 or less minutes, 20 or less minutes, 15 or less minutes, 10 or less minutes, 5 or less minutes, or 2 or less minutes. A sample can be contacted by 1 pg/ml or less, 2 pg/m or less, 4 pg/ml or less, 8 pg/ml or less, 10 pg/ml or less, 20 pg/ml or less, 30 pg/ml or less, 50 pg/ml or less, or 100 pg/ml or less proteinase K. A sample can be contacted by proteinase K at a temperature ranging from 2°C to 55°C, where specific ranges of interest include, but are not limited to: 50 to 54°C, 40 to 44°C, 35 to 39°C, 28 to 32°C, 20 to 26°C, and 0 to 6°C. In some instances a sample can be contacted by proteinase K at a temperature of 4°C, room temperature (22-25°C). 30°C, 37°C, 42°C. or 52°C. In some embodiments, a sample is not contacted with an enzymatic permeabilization reagent. In some embodiments, a sample is not contacted with proteinase K. Contact of an intact tissue with at least a fixation reagent and a permeabilization reagent results in the production of a fixed and permeabilized tissue.

IV. Sequencing

[0083] “Sequencing” or any grammatical equivalent as used herein may refer to a method used to determine the nucleotide sequence of a target nucleic acid polymer. The sequencing technique may include, for example, Next Generation Sequencing (NGS), Deep Sequencing, mass spectrometry-based sequence or length analysis, or DNA fragment sequence or length analysis by gel electrophoresis or capillary electrophoresis. Compatible sequencing techniques may be used including single-molecule real-time sequencing (Pacific Biosciences), Ion semiconductor (Ion Torrent sequencing), pyrosequencing (454), sequencing by synthesis (Illumina), sequencing by ligation (SOLiD sequencing), chain termination (Sanger sequencing). Nanopore DNA sequencing (Oxford Nanosciences Technologies), Helicos single molecule sequencing (Helicos Inc.), sequencing with mass spectrometry, DNA nanoball sequencing, sequencing by hybridization, and tunneling currents DNA sequencing.

[0084]As used herein, “next generation sequencing” refers to high-throughput sequencing methods that allow the sequencing of millions to billions of molecules in parallel. Examples of next generation sequencing methods include sequencing by synthesis, sequencing by ligation, sequencing by hybridization, polony sequencing, ion semiconductor sequencing, and pyrosequencing. By attaching primers to a solid substrate and a complementary sequence to a nucleic acid molecule, a nucleic acid molecule can be hybridized to the solid substrate via the primer and then multiple copies can be generated in a discrete area on the solid substrate by using polymerase to amplify (these groupings are sometimes referred to as polymerase colonies or polonies). Consequently, during the sequencing process, a nucleotide at a particular position can be sequenced multiple times (e.g., hundreds or thousands of times) — this depth of coverage is referred to as “deep sequencing.” Examples of high throughput nucleic acid sequencing technology include platforms provided by Illumina, BGI, Qiagen. ThermoFisher, and Roche, including formats such as parallel bead arrays, sequencing by synthesis, sequencing by ligation, capillary electrophoresis, electronic microchips, “biochips,” microarrays, parallel microchips, and single-molecule arrays.

[0085]General sequencing methods known in the art, such as sequencing by extension with reversible terminators, fluorescent in situ sequencing (FISSEQ), pyrosequencing, massively parallel signature sequencing (MPSS), expansion sequencing” (ExSEQ) (U.S. Patent No. 10,059,990), and the like are suitable for use in the methods of the invention. Reversible termination methods use step-wise sequencing-by-synthesis biochemistry that is coupled with reversible termination and removable fluorescence.

[0086]Fluorescent in situ sequencing (FISSEQ) can refer to a method to detect or sequence 3-dimensionally arranged targets in situ within a matrix, wherein the detection signal is a fluorescent signal. More specifically, FISSEQ is a method whereby DNA is extended by adding a single type of fluorescently-labelled nucleotide triphosphate to the reaction; washing away unincorporated nucleotide, detecting incorporation of the nucleotide by measuring fluorescence, and repeating the cycle. At each cycle, the fluorescence from previous cycles is bleached or digitally subtracted or the fluorophore is cleaved from the nucleotide and washed away. FISSEQ is described, for example in. Lee et al., Science. 343, 1360-3 (2014). Sequencing methods that can be employed by FISSEQ can be sequencing-by- synthesis, sequencing by ligation, or sequencing by hybridization. The targets detected or sequenced in FISSEQ can be a biomolecule of interest or a probe bound to the biomolecule of interest.

[0087]Pyrosequencing is a method in which the pyrophosphate (PPi) released during each nucleotide incorporation event (i.e., when a nucleotide is added to a growing polynucleotide sequence). The PPi released in the DNA polymerase-catalyzed reaction is detected by ATP sulfurylase and luciferase in a coupled reaction which can be visibly detected. The added nucleotides are continuously degraded by a nucleotide-degrading enzyme. After the first added nucleotide has been degraded, the next nucleotide can be added. As this procedure is repeated, longer stretches of the template sequence are deduced. Pyrosequencing is described further in Ronaghi et al. (1998) Science 281:363.

[0088]MPSS utilizes ligation-based DNA sequencing simultaneously. A mixture of labelled adaptors comprising all possible overhangs is annealed to a target sequence of four nucleotides. The label is detected upon successful ligation of an adaptor. A restriction enzyme is then used to cleave the DNA template to expose the next four bases. MPSS is described further in Brenner et al. (2000) Nat. Biotech. 18:630.

[0089]In particular embodiments, LISH Lock’n’Roll can be used in the methods of the present invention. More specifically, hybridization of probes in a sample, followed by in situ ligation (“‘LISH”), locks specifically circularized probe set around an RNA or ssDNA target sequence. Rolling circle amplification (“LISH-Lock’n’Roll”), followed by fluorescently labeled detector probe hybridization, enables simultaneous in situ quantification and localization of RNA sequences with subcellular precision. See U.S. Patent Application No. 17/790,293, entitled “In Situ RNA Analysis Using Probe Pair Ligation (Publication No. 2023/0039899) and International PCT Patent Application No. PCT/US2023/024735, entitled “In Situ Nucleic Acid Analysis Using Probe Pair Ligation.,” which claims priority to U.S. Provisional Patent Application No. 63/349,787.

[0090]Without further elaboration, it is believed that one skilled in the art, using the preceding description, can utilize the present invention to the fullest extent. The following examples are illustrative only, and not limiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES

[0091]The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and/or methods described and claimed herein are made and evaluated, and are intended to be purely illustrative and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g.. amounts, temperature, etc.) but some errors and deviations should be accounted for herein. Unless indicated otherwise, parts are parts by weight, temperature is in degrees Celsius or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.

EXAMPLE 1: BARCODING INTRACELLULAR REVERSE TRANSCRIPTION ENABLES HIGH-THROUGHPUT PHENOTYPE-COUPLED T CELL RECEPTOR ANALYSES Materials and Methods [0092]Peptide Stimulation of PBMCs. PBMCs were rapidly thawed at 37°C and added dropwise to a 15mL conical tube containing 1 mL of 37°C RPMI (ThermoFisher, Cat. No. 11875093). 6 mL of 37°C RPMI was then added slowly to w ash the cells, followed by centrifugation at 1200 rpm for 10 min. The supernatant was discarded, and cells were resuspended in 5 mL 37°C complete media (RPMI + IX BME (Thermofisher, Cat. No. 21985023 + IX GlutaMAX (Thermofisher, Cat. No. 35050061) + IX antibiotic antimycotic solution (MilliporeSigma, Cat. No. A5955) + sterile-filtered, heat inactivated human AB serum at 10% v/v (MilliporeSigma, Cat. No. H3667)) and centrifuged at 1200xrpm for 10 min to wash. Repeated for a total of two washes. The supernatant was discarded, and cells were resuspended in 10 mL complete media (IxlO⁶ cells/mL) and transferred to a 10 cm dish to rest for at least 3 hours at 37°C and 5% CO2.

[0093]After resting, 200 pL PBMCs were plated at 5xl0⁶ cells/mL per well, with 100IU IL2/mL (PEPROTECH, Cat. No., 200-02) and 2 ug peptide/mL in a 96 well V-bottom plate (Coming, Cat. No. 3894). Mutant KRAS 21-mer synthetic long peptides with the following mutations were used for stimulations: G12C, G12V, G12D, G12A. Positive control EBV peptide and KRAS WT peptide were used at the same concentration. 1 pL DMSO/mL (matching the volume of peptide added) was used for the unstimulated (no peptide) condition. Cells were incubated at 37°C and 5% CChfor ~72 hours. r0094]Cell Staining. 200 pL of Jurkat cells (to be used as a spike-in post cell-sorting) were added to 1 well of the V-bottom plate (Coming, Cat. No. 3984) at 5xl0⁶ cells/mL to be stained along with stimulated PBMC samples. The V-bottom plate containing all samples was then centrifuged at 400xg for 10 min to pellet cells, and the supernatant was discarded by gently flicking the plate. To wash, cells were resuspended with 200 pL of IX PBS -Ca-Mg (Coming, Cat. No. 21-040-CV) plus 0.05U/pL of Protector RNase Inhibitor (Roche, Millipore Sigma. Cat. No. 333540200) (PBS-RI) and centrifuged at 400xg. This was repeated for a total of tw o washes. Cells were then resuspended at 1 : 1000 Zombie green live/dead stain (BioLegend, Cat. No. 423111) in 50 pL PBS-RI for 20 min at room temperature in the dark. Cells were then washed with 150 pL PBS-RI and centrifuged at 400xg for 10 min. The supernatant was discarded, and cells were resuspended in 55 pL Brilliant Stain Buffer (BD, Cat. No. 563794) with 0.2U/pL of Protector RNase Inhibitor, for 15 minutes at room temperature in the dark w ith the following antibody dilutions: 1 : 100 CD4 RPA-T4 clone in BV605 (BD, Cat. No. 562658), 1 :250 CD8 RPA-T8 clone in AF700 (BD Cat. No. 561453), 1:50 CD3 clone UCHT1 in AF647 (BD, Cat. No. 557706), 1 :500 CD69 clone FN50 in BV786 (BD, Cat. No. 563834). and 1: 100 CD137 clone 4B4-1 in BV650 (BD, Cat. No. 564091). Cells were washed with 150 pL PBS-RI and centrifuged at 400xg for 7 min. Three additional washes were performed with 200 pL PBS-RI and centrifugation at 400xg for 7 min. Cell pellets were resuspended in 35 pL of PBS-RI. rOO95]Cell fixation with methanol. 160 pL of 100% ice-cold molecular biology' grade methanol was added dropwise to each well (final 80% v/v methanol). Cells were then mixed by gentle pipetting up and down after all methanol addition. The V-bottom plate was placed at 4°C for 15 min in the dark. 3 pL of 5% Triton-XlOO in nuclease-free water was added to each well to augment cell pelleting, and the plate was centrifuged at 400xg for 10 min at 4°C. Cells were subsequently washed two more times with 200 pL of 0.001% Triton-XlOO in PBS-RI and 400xg centrifugations for 7 min at 4°C. Stained and fixed cells were then resuspended in 10 pL of PBS-RI.

[00961INCERTS reverse transcription (RT). Each well of methanol-fixed cells was pre-annealed with primer for 1 hour at room temperature, in the dark as follows: 10 pL of cells in PBS-RI with 2 pL of 2uM well-specific barcode primer, 8 pL of 5X buffer from SuperScript IV First-Strand Synthesis System kit (ThermoFisher, Cat. No. 18091050), 5 pL of 1 : 10 diluted Protector RNase Inhibitor and 15 pL of nuclease free water. After preannealing, 150 pL of 0.001% Triton-XlOO in PBS-RI was added to cells. The plate was centrifuged at 400xg for 7 min 4°C. Supernatant was removed and cells were washed once more with 200 pL of 0.001% Triton-XlOO in PBS-RI followed by centrifugation at 400xg for 7 min at 4°C.

[0097]Cell pellets were resuspended with SSIV reverse transcription master mix containing 4 pL 5X RT buffer, 1 pL lOmM dNTP mix, 1 pL 0. IM DTT, 1 pL RNase inhibitor, 1 pL SSIV reverse transcriptase, 0.13 pL Protector RNase inhibitor, and 10.9 pL nuclease free water. Samples were transferred to 200 pL thin-walled PCR tubes and incubated at 50°C for 10 min for reverse transcription. After RT, cells were transferred back to the V-bottom plate. 175 pL of 0.001% Triton-XlOO in PBS-RI was added and cells were centrifuged at 400xg for 7 min at room temperature. Supernatant was removed and the plate was washed once more in 200 pL 0.001% Triton-XlOO in PBS-RI and centrifuged at 400xg for 7 min at room temperature.

[0098]DSP cross-linking. Cell pellets were resuspended in 200 pL of 0.25mg/mL Dithiobis (succinimidyl propionate) (DSP) (ThermoFisher, Cat. No. 22585) in IX PBS with 0.2U/pL of Protector RNase Inhibitor for 30 min at room temperature in the dark. 5 pL of IM Tris (pH 7.5) was added to each well to quench reactions at a final concentration of 20 mM Tris for 10 min. The plate was then centrifuged at 400xg for 7 min at room temperature and supernatant was discarded. Cells were washed twice with 200 pL of 0.001% Triton- Xi 00 in PBS-RI and centrifuged at 400xg for 10 min, discarding supernatant each time. The Jurkat cells were resuspended in 200 pL of PBS-RI, transferred to a 1.5 mL DNA LoBind tube, and stored at 4°C until after FACS of PBMCs. Remaining cell pellets were resuspended in 50 pL of PBS-RI each. All cells were then transferred into 1 tube for FACS analysis.

[0099]Cell-sorting and cDNA extraction. Sorting was performed on a FACS Aria™ Fusion (4-way sort) into activated and not activated populations for both CD4 (CD3⁺CD4⁺) and CD8 (CD3 CD8 ) cells. The activated population was defined as CD69⁺CD137⁺ ’ and CD69^+/ CD137⁺. The not activated population was CD69'CD137‘. A negative control (no peptide) sample was used to determine gating. Samples were sorted into PBS-RI in 1.5mL DNA LoBind tubes. After the sort, the volumes of inactivated populations were brought up to 500pL by adding the necessary volume of PBS-RI. and the volumes of activated populations were brought up to 200pL. 10000 Jurkat cells stored after DSP cross-linking were added to each tube as a monoclonal spike-in. All tubes were centrifuged at 400xg for 10 min at room temperature. Supernatant was removed from each tube such that lOOpL was remaining, to preserve the cell pellet. All samples were incubated in 50mM of DL- Dithiothreitol (DTT) (Sigma- Aldrich, Cat. No. D9163) (5.25 pL of IM DTT to lOOpL of sample) for 10 min at room temperature to reverse the DSP crosslinks. lOOpL of DNA/RNA Lysis Buffer was added to each tube containing 100 pL of sample (1:1 ratio of lysis buffer to sample) and purification proceeded as per Jt/zcZr-DNA/RNA Microprep Plus kit instructions (Zymo. Cat. No. D7005). Samples were eluted in 22pL of nuclease free water and stored at - 20°C until PCR amplification.

Amplification and Sequencing. 8pL of cDNA from each sorted population was amplified via 20 cycles of PCR using the KAPA2G Fast Multiplex Kit (Roche. Cat. No. 07961430001) and the 1MM FR3AK-Seq multiplex primer set,¹¹ with each forward primer at 0.00835 uM and the reverse primer at 0.125 uM. PCR conditions were as follows: 1) 95°C for 3 min, 2) 95°C for 15 sec, 47°C for 30 sec, and 72°C for 30 sec for 20 cycles. 3) 72°C for 1 min. A second PCR was performed to add cell subpopulation-barcoded forward and reverse primers using 2 pL of PCR1 product and primers at 0.25 uM each with the Herculase Fusion DNA Polymerase kit (Agilent, Cat. No. 600677). PCR conditions were as follows: 1) 95°C for 2 min, 2) 95°C for 20 sec, 58°C for 20 sec, and 72°C for 30 sec for 20 cycles. 3) 72°C for 3 min. Barcoded samples were then mixed together and an equal volume of sparQ PureMag beads (Quantabio, Cat. No. 95196-005) were added. Bead clean-up proceeded as per manufacturer’s instructions. Briefly, samples and beads were mixed together until homogenous, incubated at room temperature for 5 min then placed on a magnet. Supernatant was removed and samples were washed twice with 80% ethanol (30 seconds each time) while still on the magnet. Beads were then dried and eluted in nuclease free water. The pooled sample was assessed on a 1% low⁷ melting point agarose (Invitrogen, Cat. No. 16520) gel in IX TAE buffer and then gel extracted per Monarch Gel Extraction kit protocol (NEB, Cat. No. T1020S). Sequencing w as performed on an Illumina NextS eq with 150bp single-end reads.

Jurkat spike-in experiment. INCERTS w as performed as described above, with the following modifications. CD14- PBMCs were stimulated for 2.5 days with human T activator CD3/CD28 Dynabeads (ThermoFisher, Cat. No. 11131D). IxlO⁶ Dynabead stimulated PBMCs were aliquoted into each of 8 wells of the 96-well V bottom plate, and 10, 100, 1000, and 10,000 Jurkat cells w ere added into tw o w ells each. Cells were not stained, but each well w as fixed with methanol as described above. The remainder of the protocol, until the sort, proceeded as described above, and each of the 8 wells received a unique barcoded TCR reverse transcription primer. During the sort, debris and doublets w ere gated out, and then all cells were collected. Post-sort, after the DTT de-crosslink step, the 1.5mL tube was heated to 95°C for cell-lysis. Lysis buffer was then added to the tube and cDNA extraction and PCR proceeded as described above.

ELISPOT. The frequency of IFNy secreting cells was assessed by the Johns Hopkins Immune Monitoring Core using an ELISpot assay (Mabtech, Cat. No. 3420-4HST) with responder PBMC at 2xl0⁵ cells per well, KRAS peptides at 2 ug/ml in DMSO (from 4 mg/ml stock), CEF (CTL, Cat. No. PA-CEF-002) and aCD3 antibody (Mabtech. in kit) at vendor recommended concentrations. Tissue culture medium (RPMI-1640) was supplemented with 10% FBS (Hyclone, Cat. No. SH30071.03). The assays were incubated overnight (37C, 5% CO2, >18 hr) and after development w ere read using an iSpot spectrum reader (AID). The results underwent technical review⁷ before release to the investigator.

In FIG. 2D. ELISPOT values were averaged across the four peptides for each donor and plotted against the average number of total productive KRAS-reactive sequences across the four peptides, identified by INCERTS for that donor. Single-cell RNA (scRNA-seq) and TCR (scTCR) sample preparation and sequencing. scRNA library preparations were performed on unstimulated PBMCs from one of the four donors, using the lOx Genomics Chromium™ Single Cell system and Chromium™ Single Cell 5' Library & Gel Bead Kit v2 (lOx Genomics, Cat. No. PN-1000263). TCRs were enriched by following manufacturer’s instructions using the TCR Amplification kit (lOx Genomics, Cat. No. PN- 1000252). The initial cell input was 17,000 PBMCs to recover a total of 10.000 cells. Sequencing, at a depth of 50,000 reads per cell, was performed on the NovaSeq platform (Illumina) using lOx Genomics recommended features.

Software. Illumina Fastq files were demultiplexed based on sample-specific and sorted population-specific barcodes using the cutAdapt⁵² software with settings allowing for 1 mismatched nucleotide in the barcode. MiXCR’³ version 3.0.13 was then used to identify TCR sequences from demultiplexed fastq sequencing files with the following settings: — species hs -starting-material ma -receptor-type TRB —5 -end v-primers —3 -end c-primers — adapters no-adapters —assemble “-OaddReadsCountOnClustering=true.” All sequences with clone count >=10 were used for further analysis. All further data processing was performed using R and Microsoft Excel. TCR sequence clustering was performed with GLIPH2³⁸ using reference version 2.0, CD48, and all amino acids interchangeable for settings. The motif diagram was generated using the msa Bioconductor package in R.

Identifying INCERTS Reactive TCR Sequences. TCR sequences were considered to be reactive to a peptide if the sequence was productive, present in the population that was positive for either or both CD69 and CD137, and was not found in CD69-CD137- population at a frequency above 1% of the total counts of the specific CDR3 amino acid clone. TCR sequences were considered to be reactive for multiple peptides if the clone count associated with that peptide was at a frequency above 1% of the total counts of the specific CDR3 amino acid clone in the corresponding activated cell population. 1% clone specific frequency cutoff was determined based on the number of Jurkat sequences that were associated with a different well-specific barcode. Although Jurkat cells received a unique TCR barcode, in the sequencing data of the activated populations, 1% of the Jurkat clone sequences were incorrectly associated with a different barcode, potentially due to chimeric amplicon formation during PCR.

Single-cell RNA and TCR-seq analysis. Sequences were processed using the Cellranger 5.0. 1 pipeline (lOx Genomics) and mapped to the human reference genome (GRCh38). The raw feature-barcode matrix was processed with Scanpy (version 1.8. 1),⁵⁴ putative cell doublets were removed using the Scrublet package,⁵’ and leiden clustering was performed at a resolution of 2.0 to capture major cell types and subtypes. Differentially- expressed genes across the leiden clusters were determined using the scanpy.ll.rank genes groups function. Clusters were manually annotated based on the RNA expression of known cell type marker genes, fdtered using Besca,⁵⁶ and confirmed using annotation pipelines including Azimuth⁵⁷ and Cellty pist.⁵⁸ TCR repertoire analysis was performed with Scirpy (version 0.9. 1)⁵⁹ and productive TCR chain pairing status was determined with the scirpy. tl.chain_qc() function. Comparisons between INCERTS TCRp CDR3 nucleotide sequences and single-cell TCRp CDR3 sequences were performed using R v4. 1. 1. TCR CDR3 nucleotide sequences were considered a match to a single-cell TCRp CDR3 nucleotide sequence if they were identical, as were J gene calls. In cases where various single-cell alpha chains were paired to the same beta chain sequence, INCERTS T cell ty pes were considered a match if at least one of the possible alpha-beta paired phenotypes was identical.

PBMCs from patients who received a novel mutant KRAS peptide vaccine are part of the on-going clinical trial NCT05013216.

Table SI (not shown). List of productive, peptide reactive TCRP sequences identified using INCERTS method. Peptide. CD4 or CD8 T cell type, and cancer ty pe corresponding to each CDR3 are listed. CDR3s reactive to more than one peptide are listed multiple times.

Table S2 (not shown). TCR chains reactive to more than one peptide.

Table S3 (not show n). GLIPH2 output of identified clusters of peptide-reactive, productive TCRP CDR3 sequences identified via INCERTS. Refer to GLIPH2 publication for column meanings. Freq column values are MiXCR clone count.

Table S4 (not shown). Single-cell and INCERTS sequence information for overlapping TCRp CDR3s. Cell types and J genes match between single-cell and INCERTS. Full-length sequence information for published KRAS-reactive TCRs.

Table S5 (not shown). Summary- of each population, the number of sorted cells, the number of unique, productive. TCR CDR3 clones identified, and sequencing quality data for each population.

Results

FIG. 1 A illustrates a typical INCERTS workflow. First, samples are stained with fluorescently conjugated antibodies to label surface proteins of interest. Stained cells are then fixed and permeabilized with methanol, which largely preserves the integrity of mRNA.¹⁰ Each sample is then combined with a TCRP reverse transcription (RT) primer containing a unique sample-specifying barcode. After RT, cells are cross-linked with dithiobis(succinimidyl propionate) (DSP) to improve intracellular retention of cDNA during sorting. Samples are then mixed and sorted into populations of interest based on their labeled surface markers. TCR[3 cDNA is next extracted from each sorted population and amplified with Framework Region 3 Amplification Sequencing (FR3AK-seq)ⁿ primers and subpopulation-specific barcodes. In this way. TCR0 sequences in the final sequencing library carry both sample-specific barcodes and cell subpopulation-specific barcodes (FIG. IB). Barcode demultiplexing then maps each TCR sequence to a unique cell population and the sample from which it originated (FIG. 1C-1D). In a test of barcode fidelity, INCERTS was performed on T cells from two different donors, which were mixed and sorted into CD4⁺ and CD8⁺ T cell subpopulations. Negligible barcode exchange between samples or cell subpopulations (FIG. 1C) was observed. These data suggest that INCERTS can be used to analyze hundreds of samples and millions of cells in a single flow sort with negligible barcode misassignment. To assess assay sensitivity and linearity, increasing numbers of clonal Jurkat T cells were spiked into a background of 1 million PBMCs (FIG. IE). Jurkat clonal frequency was linearly proportional to cellular input (linear regression R² = 0.92 and p-value = 0.0001). At the lowest level of 10 spiked-in Jurkat cells, the Jurkat CDR3 sequence was still detectable above background. The top two most frequent PBMC T cell clonal frequencies were constant across the Jurkat titration.

There is rapidly growing interest in the discovery of typically rare tumor-specific TCRs for therapeutic applications, and INCERTS is designed to screen the millions of cells from multiple samples required for this type of analysis. Therefore, the present inventors assessed the utility of INCERTS for identifying tumor-reactive TCRs directly from patients receiving a novel mutant KRAS peptide vaccine. Mutated KRAS makes up about 85% of all RAS mutations and has been previously associated with clonal expansion of anti-tumor T cells in human patients. RAS mutations are found in approximately 30% of all cancers, and in over 90% in pancreatic cancers.^{12 17} Activating KRAS mutations are highly conserved and tend to occur at restricted amino acid positions. TCRs that recognize dominant KRAS mutants, whether to endogenous protein or peptide vaccine,¹⁸'²⁰ are thus potential candidates for cellular therapies.^17-25 The present inventors therefore used INCERTS to identify KRAS- reactive TCRs from individuals with pancreatic or colorectal cancer who were immunized with a mutant KRAS peptide vaccine (NCT04117087). The mutant KRAS peptide vaccine comprises a mixture of six 21 amino acid long synthetic peptides encompassing common KRAS hotspot mutations in pancretic and colorectal cancer - five alterations at the G12 position and one alteration at the G13 position. The dosing regimen includes both prime and boost phases, with concomitant anti-PD-1 or anti-CTLA4 immune checkpoint inhibitor therapy (FIG. 2A). Peripheral blood draws were performed at the indicated timepoints. Samples from timepoints at which T cells demonstrated peptide-reactivity by IFNy ELISPOT were prioritized for INCERTS analysis. PBMCs from each of the four donors (two with pancreatic cancer and two with colorectal cancer) were split into six separate peptide stimulation cultures each: four with individual mutant KRAS peptides (four of six mutant KRAS peptides were used due to limited sample availability, of which 3 corresponded to the most reactive peptides by ELISPOT (G12C, G12V, G12A) and one corresponded to the most reported TCR target, G12D), one with wild- tjpe (WT) KRAS peptide, one with EBV peptide, and one negative control culture without any peptide stimulation (FIG. 2B). After 72 hours, the 28 cultures were stained for CD3, CD4. and CD8 as well as the T cell activation markers CD69 and CD 137. Cells were then methanol fixed, reverse transcribed, and DSP cross-linked as outlined in FIG. 1 A. The 28 samples were then mixed for a single FACS separation into four cell subpopulations: CD4⁺ or CD8⁺ T cells that were either activated (peptide-reactive) or not activated based on CD69 and CD137 positivity (FIG. 2B). cDNA was subsequently extracted from these four phenotypic populations, PCR amplified using FR3AK-seq primers and phenotypic barcodes.¹¹ and then sequenced on a single Illumina flow cell.

Overall, INCERTS identified 186 productive, peptide-reactive TCRp clones from the 28 stimulation conditions. 119 (64%) were mutant KRAS peptide reactive (Table SI), 24 (13%) were WT KRAS peptide reactive, and 43 (23%) were EBV reactive. Of these 186 reactive TCR sequences, 114 were from 8,206 activated CD4⁺ T cells and 72 of were from 7,170 activated CD8⁺ T cells (Table S5), in line with the ability of synthetic long peptides to induce both robust CD4⁺ and CD8⁺ T cell responses.³⁰'³³ From among the 349,713 not activated CD4⁺ T cells and the 73.269 not activated CD8⁺ T cells, 2,242 and 866 unique, productive TCRp sequences, respectively, were also identified (Table S5).

Fifteen TCRp clones were reactive to at least 2 peptides (Table S2) - ten from CD4⁺ sorted T cell populations and five from CD8⁺ sorted T cell populations. Five of these fifteen TCRp clones were reactive against multiple mutant KRAS peptides. These five multi- reactive sequences (four from CD4⁺ cells and one from CD8⁺ cells) may have greater clinical potential. Although CD8⁺ T cell-derived TCRs are most commonly nominated as therapeutic candidates. CD4⁺ T cell derived TCRs are increasingly proposed as viable therapeutic candidates.³⁵'³⁷ Tn comparison, four of the fifteen cross-reactive TCRp clones appeared to be reactive to both WT and mutant KRAS peptides, thus limiting their therapeutic potential.

Analysis of mutant KRAS peptide-reactive TCRPs using the GLIPH2 software³⁸ identified 21 convergent clusters. The most statistically significant cluster featured a conserved motif with N-terminal CLCASSL and C-terminal QYF (FIG. 2C). CASSL with QYF. is also found in published KRAS-reactive TCRPs (Table S4).¹⁸-²¹-²⁵-²⁸ Interestingly, this motif was detected in sequences from both CD4⁺ and CD8⁺ T cell populations from two different donors. Although CD4⁺ and CD8⁺ TCRPs are typically non-overlapping due to their distinct restrictions, studies have reported sequence similarities in CD4⁺ and CD8⁺ TCRPs in autoimmune,³⁹ viral,⁴⁰ and cancer⁴¹ patient populations. Notably, one sequence in this cluster is reactive to the control EBV peptide, and another sequence in this cluster is reactive to the WT KRAS peptide. Furthermore, all clusters containing TCRp clones reactive to multiple mutant KRAS peptides also contained an EBV or WT KRAS reactive TCRp clone, suggesting that these TCRs may be promiscuous in antigen specificity (Table S3). Because PBMC timepoints were selected for INCERTS analysis based on T cell reactivity to mutant KRAS by IFNy ELISPOT, we next examined ELISPOT concordance with the INCERTS findings. The present inventors found that donors with more mutant KRAS peptide-reactive T cells by ELISPOT also had more mutant KRAS-reactive TCRs detected via INCERTS (FIG. 2D) (R² = 0.94, p-value = 0.03).

PBMCs from donor 12 were also analyzed using combined scRNA-seq and TCR-seq (10X Genomics). The present inventors therefore queried this complementary dataset to find TCRP CDR3 sequences that matched INCERTS-identified sequences. Altogether 65 matching TCRp CDR3 nucleotide sequences were found. Of these, 56 (86.2%) had a TCRcc chain that was sufficiently defined for functional reconstruction of the TCR. Notably, all 65 (100%) J gene sequences matched between the two data sets, as expected based on the location of the reverse transcription and FR3AK-seq primers used in INCERTS analysis. Furthermore, 53 of the 65 overlapping TCRPs (81.5%) were identically annotated as CD4⁺ or CD8⁺ in both datasets (FIG. 2E). Five of the 65 overlapping TCRPs were identified by INCERTS as potentially mutant KRAS-reactive (Table S4). Of these five sequences, one TCR with TRBV28 and TRBJ2-7 shares the same V/J pairing with a previously published mutant KRAS-reactive TCR.²⁸ Another TCR with TRAV4 and TRAJ15 overlaps with V and J genes reported for four published mutant KRAS-reactive sequences (Table S4).²¹ Because the scRNA-seq data was generated from whole PBMCs, the present inventors expect that enrichment for T cells upstream of scRNA-seq would increase the number of overlapping TCRPs available for comparison. Indeed, these findings underscore the complementary nature of INCERTS and scRNA-seq, as the former enables identification of many candidate mutant KRAS-reactive TCRs, while the latter provides detailed phenotypic information and paired alpha/beta TCR sequences. Overall, these findings demonstrate the accuracy and utility of INCERTS to identify potentially neoantigen-specific TCRs. When integrated with patient-matched scRNA-seq data, these TCRs may be fully reconstructed for functional validation.

Discussion

Fewer than 50 KRAS-specific TCRs have been reported in the literature to date.^18,21,25’²⁸’²⁹’⁴² However, using INCERTS, 119 candidate mutant KRAS-reactive TCRPs, none of which overlap with known TCRs. were identified in a single experiment. Of these, 25 were reactive to G12C, 26 to G12V, 47 to G12D, and 21 to G12A. Notably, TCRPs specific for G12A are not yet reported in the literature, while G12V and G12D-specific TCRPs are most common. Additionally, only 1 of the established mutant KRAS-reactive TCR sequences was obtained from a pancreatic cancer patient, while the present inventors report 38 candidates from two pancreatic cancer patients. The present inventors further report 81 candidate KRAS-reactive TCRPs from two colorectal cancer patients. Additionally, a shared motif (beta chains) and shared V/J usage (alpha and beta chains) was identified between the candidate mutant KRAS-reactive TCRs and published mutant KRAS- reactive TCRs.

Other methods have been developed to define antigen-specific TCRs linked to cellular phenotypes. Traditionally, antigen-specific populations have been identified by activation marker positive populations, multimer positivity, or CFSE dilution followed by bulk RNA- seq of individual populations.⁴⁴ One such approach, MIRA⁴⁵, enables identification of TCRs specific for many antigens across many samples. However, each stimulation condition requires sorting into multiple populations, limiting the method's scalability. An alternative approach, MANAFEST⁴⁶, relies on expansion of antigen-specific T cells that are quantified by bulk-sequencing. A notable limitation of this approach is the inability⁷ to link TCRs with cellular phenotype. INCERTS ’s intracellular barcoding strategy can be incorporated into the experimental setup of assays such as MIRA or FEST, increasing the number of antigen stimulations and samples that can be processed simultaneously and efficiently. More recent methods for identifying TCR antigen specificity utilize scRNA-seq, enabling simultaneous detection of phenotype and paired alpha-beta TCRs. For example, SELECT-seq⁴⁷ is a method that sorts single T cells after antigen stimulation. TCRs are sequenced to identify expanded clones, which are then analyzed individually via scRNA-seq. Although this method reduces the cost of sequencing by limiting transcriptomic analysis to clones of interest, the total number of T cells examined is limited to hundreds to low thousands. Another recently developed technology, TetTCR-Seq HD,⁴⁸ sorts on tetramer positive populations prior to scRNA-seq. While tetramer-positive populations reveal antigen-specific TCRs, this approach relies on pre-defined epitopes. Furthermore, the use of tetramers requires a high-affinity TCR interaction, which may miss important lower-affinity TCRs.⁴⁹ Other methods successfully linking protein-level phenotypic and transcriptomic information from immune cells, including CITE-seq,⁸ INs-Seq⁵⁰ and CLInt-seq⁵¹, have also been reported. Each of these use scRNA-seq for readout, making them expensive and constrained in the number of antigen-reactive clones assessed per experiment.

Overall, INCERTS enables sample mixing for efficient cell sorting and phenotype- embedded repertoire sequencing. As described herein, the present inventors used INCERTS to discover over 100 candidate mutant KRAS-reactive TCR sequences from a set of patients receiving a mutant KRAS peptide vaccine. Quick, inexpensive INCERTS-based quantification of specific TCRs or motif-containing TCRs across numerous patient samples and/or over multiple timepoints may also be used to track specific immune responses. Additionally, INCERTS can be applied for comparison of repertoires from stimulated and sorted populations to repertoires from millions of unstimulated and unsorted cells for direct quantification of clonal enrichment. In further embodiments, the method may be readily adapted to any markers of interest (e.g., to memory populations, exhausted populations, etc.). In other embodiments, INCERTS could also be applied to B cells to identify BCR sequences in populations of interest (e.g., defined by phenotypic markers or even antigen-bound BCRs). Importantly, INCERTS may be extended beyond immune cells and their antigen specific receptors to identify differences in cell populations with and without specific sequence variants or transcriptional patterns. INCERTS is thus a simple approach to link proteomic and transcriptomic information from hundreds of samples comprising millions of cells in a single integrated workflow.

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EXAMPLE 2: IN SITU INCERTS FOR SPATIAL ANALYSIS OF RNA SEQUENCES

In this Example, In Situ INCERTS is applied to the spatial localization of immune receptor sequences (TCRs). In Situ INCERTS for TCR analysis works by barcoding individual TCR cDNA molecules during reverse transcription of TCR mRNA, such that the barcodes can be determined in situ and then used as the key to spatially map TCR sequences determined via ex situ sequencing of the cDNA molecules that include both the spatial barcode and the TCR sequence. As described herein, the present inventors adapted the INCERTS protocol to fixed cells adhered on a slide. However, this protocol can also be applied to fixed tissue sections, including FFPE. The present inventors developed the protocol using monoclonal T cell (Jurkat) and B cell (Ramos) lines either separately or mixed together. Although other embodiments can involve in situ sequencing, in this Example the present inventors employed LISH-LnR to spatially map the RT primer-associated spatial barcodes. A more detailed depiction of the LISH-LnR probe configuration and workflow are provided in FIGS. 5-6.

In particular embodiments, In Situ INCERTS comprises the following main steps (FIG. 3): 1) cell adherence, 2) cell fixation and permeabilization, 3) INCERTS, 4) barcode amplification via LISH-LnR, 5) spatial analysis of the amplified barcode, and 6) ex situ analysis of the immune receptor cDNA. Importantly. LISH-LnR, cDNA barcodes could also be determined using any of the various in situ sequencing technologies that have been developed.

Experimental Protocol:

Step 1 - Cell Adherence. 20 pL of Jurkat cells in IX PBS, at a concentration of 10xl0⁶ cells/mL, are pipetted onto a 10mm cover slip coated in poly-D-lysine, and allowed to settle at 37C for at least 20 minutes. 500pL of PBS is added to the cover slip, to wash away cells that are not adhered, and then discarded. The wash is repeated.

Step 2 - Cell Fixation and permeabilization. Cells are fixed by adding 500 pL of 4% PFA (or 10% formalin) at room temperature for 15min. Fixative is removed and 500 pL of IX PBS-RI (PBS with Protector RNase Inhibitor added at a final concentration of 0.05 U/pL) is added to the cover slip as a wash. The PBS is discarded, and the wash is repeated twice, for a total of 3 washes. It’s important to note that while the INCERTS protocol uses methanol to simultaneously fix and permeabilize cells, because tissue slides are likely to be fixed with formalin, this part of the protocol was changed to formalin with that long-term application in mind. For permeabilization, 250 pL of 0.1% Triton X-100 in 1X-PBS (with Protector RNase Inhibitor at final concentration of 100 U/mL and Superase-In Inhibitor at final concentration of 200 U/mL) is added to the cells for 20 min at room temperature. Permeabilization buffer is removed, and the slide is washed three times with 250 pL IX PBS- RI as previously described. Cells are then incubated with 250 pL 0. IN HC1 (with Protector RNase Inhibitor at final concentration of 100 U/mL and Superase-In Inhibitor at final concentration of 200 U/mL) for 10 minutes at room temperature. HC1 solution is removed and cells are washed three times with 250 pL IX PBS-RI. Step 3 - INCERTS. Prior to primer annealing, a buffer exchange is performed where 200 pL of 2X SSC| 10% formamide is added to the cells and then removed. Although INCERTS used RT buffer for primer annealing, SSC is used as a hybridization buffer in situ techniques, and formamide enhances primer specificity, thus the switch was made to 2X SSC| 10% formamide for the primer annealing step. Each slide is placed cell side down onto a 50 pL droplet (cells in contact with droplet), where the 50 pL consisted of 41.25 pL of 2X SSC| 10% formamide, 2.5 pL of 2 pL RT primer, and 6.25 pL of Protector RNase Inhibitor at 2 U/pL. Cells are incubated with this primer mixture for 1 hour, at room temperature. Excess primer annealing liquid is removed by holding the slide vertically with forceps and tapping gently so excess liquid drains onto a kimwipe. Cells are then w ashed by placing the slide cell-side down onto a 100 pL droplet of 2X SSC for a few seconds, and then tapping onto a kimwipe. This wash is repeated. Another buffer exchange is performed in IX RT buffer, where slides are placed cell-side down into a 50 pL droplet of IX RT buffer. Excess liquid is tapped onto a kimwipe, and then cells are incubated in 50 pL consisting of: 10 pL 5X RT buffer, 2.5 pL lOmM dNTP mix, 2.5 pL 0.1M DTE. 2.5 pL RNase inhibitor, 2.5 pL SSIV reverse transcriptase, 0.31 pL Protector RNase inhibitor, and 29.69 pL nuclease free water, for 10 min at 50°C, for reverse transcription. Cells are then washed in 0. IX SSC with O. lU/pL Protector RNase Inhibitor (SSC-RI) by placing the slide cell-side down onto 100 pL of solution and then tapping the slide onto a kimwipe as previously described. The wash is repeated tw o more times, for a total of three times.

If the cDNA from the slide is to be subsequently used for qPCR or sequencing, an RNase H release step is performed. RNase H destroys the RNA strand of RNA-cDNA hybrid helices. 50 pL of the following RNaseH solution was placed directly onto the cell-side of the slide: 2.5 pL RNaseH. 37.5 NFW, 2.5pL 0. IM DTT. and 10 pL 5X RT buffer. Cells are incubated at 37°C for 20 minutes, and then as much of the 50 pL volume that could be pipetted off the slide is stored at -20°C until qPCR was performed. If qPCR is not required, slides are stored overnight at 4°C, where slides are cell-side down on 50 pL of 2X SSC with 0.04 U/pL of Protector RNase Inhibitor, until Lock ‘n’ Roll is performed the next day.

Steps 4-5 - Ligation In Situ Hybridization Lock’n’Roll (LISH-LnR). The cell-side of slides are placed onto a 100 pL droplet of 2X SSC| 10% formamide as a buffer exchange. Excess liquid from the slide is tapped onto a kimwipe, and then the cell-side of slides are placed face down onto a 50 pL droplet of 200nM ligation:blunt bridge hybridization (1 :2) Mix (IpL of 100 uM LnR_5P specific to Jurkat, IpL of 100 uM LnR_3P specific to Jurkat, 1 pL of 100 uM LnR_5P specific to Ramos, IpL of 100 uM LnR_3P specific to Ramos, 3 pL of each 100 uM Blunt Bridge Probe (3P and 5P), 100 pL Formamide, 300 pL 20X SSC . 10 pL Protector RNAse Inhibitor and 583 pL H2O) for Ihr at 45°C in a humid chamber. Cells are then washed by placing the cell-side onto a 100 pL droplet of 2X-SSC, then tapping excess liquid onto a kimwipe. The wash is repeated two additional times, for a total of 3 washes.

Cells then undergo a buffer exchange by placing the cell-side onto a 50 pL droplet of IX T4 buffer. Slides are placed cell-side down onto 50 pL of a T4 DNA ligation mixture (2 pL T4 DNA ligase (High Concentration), 50 pL 10X rxn buffer, 448 pL NFW, and 1 pL Protector RNase inhibitor), and incubated for 1 hour at 30°C in a humid chamber. This step uses the bridge probe to circularize by ligation the two LnR binding probes which have annealed adjacently on the RT primer’s barcode. Cells are washed three times with 100 pL of 2X SSC as previously described.

Cells then undergo a buffer exchange with 50 pL of lX-Phi29 reaction buffer. Excess liquid is tapped off onto a kimwipe and then slides are placed cell-side down onto a 50 pL droplet of Phi29 reaction mix (50 pL of lOx Phi29 Buffer, 5 pL of lOOmM dNTP mix , lOOpL Phi29, and 345 pL NFW). Phi29 generates a rolling circle product, thereby amplifying the barcode sequence, along with readout probe binding sites that are affixed to the LISH-LnR probe sets. Cells are then washed three times with 2X SSC as previously described.

Step 6 - Spatial barcode imaging and cDNA sequence analysis. For ex situ analysis of the TCR sequence, cDNA can be released and collected at this point using RNaseH. The TCR cDNA will have both the spatial barcode and TCR sequence linked on the same molecule. Standard Illumina or long read sequencing techniques can be used to determine the barcode-TCR association. Meanwhile, the rolling circle amplified barcode and readout probe sequences (Phi29 rolling circle amplicon) will remain in situ, sterically trapped within the fixed tissue but available for spatial analysis. An alternative workflow would release the cDNA after the spatial analysis of the rolling circle amplicon.

In this Example to detect specific LISH-LnR products, cells are incubated with 50 pL of detector probes (20 nM) in 2X SSC with Protector RNase Inhibitor (1%) (10 pL each probe, 50 pL 20X SSC, 410 pL NFW and 0.5 pL Protector) for 30 min at room temperature in the dark. Cells are washed three times with 2X SSC as previously described. After the last wash, slides are gently tapped to remove as much excess liquid as possible for mounting. All slides are mounted onto one drop of Prolong Glass Antifade and to cure overnight at room temperature in the dark.

Imaging is performed on a FISHScope (Olympus IX 83 inverted microscope with a Lumencor SPECTRA-III 360 light engine). 60X magnification is used to obtain a z-stack of images for analysis (spot quantification). Analy sis is performed using Nikon Elements software.

Results

When assaying Jurkat cells with the Jurkat RT primers, many spots in each cell are visualized (FIG. 4A), demonstrating that LISH-LnR detection of cDNA barcodes succeeded. The generation of Jurkat TCR cDNA was additionally confirmed via qPCR validation. Because the LnR probes target the barcoded primer, rather than the cDNA product, spots are still detected in the condition without Superscript IV RT enzyme addition (FIG. 4B). In negative control conditions where 1) no RT primer is added (FIG. 4C) and thus there is no LnR template, and 2) no T4 DNA ligase is added (FIG. 4D), preventing the generation of the rolling circle amplicon (RCA), no spots are observed.

The present inventors also performed the in situ protocol on an additional cell line, a monoclonal B cell line, Ramos cells, because a long-term goal of this protocol would be to apply it to B cells in tissue as well. The present inventors used Ramos RT primer on Ramos cells (FIG. 4E), and observe spots as expected, although fewer spots per cell are observed compared to the Jurkat cell line. To test primer specificity, a mixed RT primer set was used on each cell line individually (both Jurkat and Ramos RT primers incubated with only Jurkat cells, and only Ramos cells) (FIG. 4F for Jurkat and FIG. 4H for Ramos). In both of these experimental conditions, spots are for both RT primers within a single cell (a zoomed-in representative cell is displayed for each cell type), likely explained by incomplete removal of unextended RT primers. When a mixture of both Jurkat and Ramos cells was plated and incubated with mixed primers, again spots were observed corresponding to both RT primers in all cells (a representative cell is shown in FIG. 4G). In additional experiments, the specificity of the RT primer annealing and removal will be increased.

It is important to note that in practice, the amplification of barcodes associated with unextended primers does not preclude the successful spatial mapping of cDNA sequences. The present inventors expect any final data set will include both spatial barcodes that are not linked to desired cDNA sequences, as well as cDNA sequences that are not linked to spatially mapped barcodes. To achieve optimal primer specificity', the stringency of the washes can be increased after primer annealing to remove nonspecifically-bound primers. While 2X SSC is used for washes after primer annealing to maximize the amount of primer available for cDNA transcription, the concentration of SSC can be decreased for a more stringent wash. Additionally, a heated wash can be used, where the temperature of the wash buffer is above the annealing temperature of the primer. Currently, a heated wash is used after cDNA generation, but it may be useful after primer annealing as well.

In further development, a mixture of barcodes are used such that each cell is represented by a unique combination of random barcodes because the multiplicity of TCR and/or BCR molecules within a single cell will be associated with a unique set of barcodes in close spatial proximity. Depending on the number of T cells the present inventors expect to see in tissues slides on average, the total number of barcodes needed to provide enough diversity such that each cell will in fact have a unique combination of barcodes will need to be estimated. The number of barcodes will also depend on the number of TCR molecules the present inventors expect to identify per cell, which can be estimated by the number of spots seen per cell, using cell lines. A more accurate estimate of the number of receptor RNA molecules per cell will likely be obtained by altering the LnR binding probe design, such that spots are only visualized spots for which cDNA was generated. Furthermore, in other embodiments designed to obtain both alpha and beta TCR information for each cell, the number of barcodes can be doubled.

Furthermore, to recover the TCR cDNA to obtain the TCR sequences, RNase H release is performed prior to imaging, to recover as much cDNA as possible. cDNA can also be collected after imaging in any number of ways. TCR or BCR cDNA will then be amplified during PCR.

While there are still optimizations remaining for the development of this In Situ INCERTS methodology, the present inventors’ preliminary data demonstrates the goal of spatially localizing TCR and BCR molecules. The protocol is easily adaptable to identity⁷ BCR sequences for each of the five main isotypes. A spatial protocol that can capture both TCR and BCR sequence information would be extremely powerful in many diseases, including tissue damaging autoimmune diseases.

References

(1) Liu. S.; lorgulescu, J. B.; Li, S.; Borji, M.; Barrera-Lopez. I. A.; Shanmugam, V.; Lyu, H.; Morriss, J. W.; Garcia, Z. N.: Murray, E.; et al. Spatial Maps of T Cell Receptors and Transcriptomes Reveal Distinct Immune Niches and Interactions in the Adaptive Immune Response. Immunity 2022, 55 (10), 1940-1952.e5.

(2) Moses, L.; Pachter. L. Museum of Spatial Transcriptomics. Nat. Methods 2022 195 2022, 19 (5), 534-546.

EXAMPLE 3: BARCODING INTRACELLULAR REVERSE TRANSCRIPTION ENABLES HIGH-THROUGHPUT ANTIBODY- ANTIGEN ASSOCIATION Materials and Methods

Cell Staining. Cells used in this assay are compnsed of a set of cells that express a set of antigens. Cells in each well are stained using a different patient serum or plasma sample containing primary' antibodies. To wash, cells are resuspended with 200 pL of IX PBS -Ca- Mg (Coming, Cat. No. 21-040-CV) plus 0.05U/pL of Protector RNase Inhibitor (Roche, Millipore Sigma. Cat. No. 333540200) (PBS-RI) and centrifuged at 400xg. This is repeated for a total of two washes. Cells are then resuspended at L 1000 Zombie green live/dead stain (BioLegend, Cat. No. 423111) in 50 pL PBS-RI for 20 min at room temperature in the dark. Cells are then washed with 150pL PBS-RI and centrifuged at 400xg for 10 min. The supernatant is discarded, and cells are resuspended in 55 pL Brilliant Stain Buffer (BD, Cat. No. 563794) with 0.2U/pL of Protector RNase Inhibitor, for 15 minutes at room temperature in the dark with secondary antibodies that recognize the patients’ antibodies of interest (e.g., human IgG). Cells are washed with 150 pL PBS-RI and centrifuged at 400xg for 7 min. Three additional washes are performed with 200 pL PBS-RI and centrifuged at 400xg for 7 min. Cell pellets are resuspended in 35 pL of PBS-RI.

Cell fixation with methanol. 160 pL of 100% ice-cold molecular biology grade methanol is added dropwise to each well (final 80% v/v methanol). Cells are then mixed by gentle pipetting up and down after all methanol addition. The V-bottom plate is placed at 4°C for 15 min in the dark. Three (3) pL of 5% Triton-XlOO in nuclease-free water is added to each well to augment cell pelleting, and the plate is centrifuged at 400xg for 10 min at 4°C. Cells are subsequently washed two more times with 200 pL of 0.001% Triton-XlOO in PBS- RI and 400xg centrifugations for 7 min at 4°C. Stained and fixed cells are then resuspended in 10 pL of PBS-RI.

INCERTS reverse transcription (RT). Each well of methanol-fixed cells is preannealed with primer for 1 hour at room temperature, in the dark as follows: 10 pL of cells in PBS-RI with 2 pL of 2uM well-specific barcode primer, 8 pL of 5X buffer from SuperScript IV First-Strand Synthesis System kit (ThermoFisher, Cat. No. 18091050), 5 pL of 1 : 10 diluted Protector RNase Inhibitor and 15 pL of nuclease free water. After pre-annealing, 150 pL of 0.001% Triton-XlOO in PBS-RI is added to cells. The plate is centrifuged at 400xg for 7 min 4°C. Supernatant is removed and cells are washed once more with 200 pL of 0.001% Triton-XlOO in PBS-RI followed by centrifugation at 400xg for 7 min at 4°C.

Cell pellets are resuspended with SSIV reverse transcription master mix containing 4 pL 5X RT buffer, 1 pL lOmM dNTP mix. 1 pL 0.1M DTT. 1 pL RNase inhibitor, 1 pL SSIV reverse transcriptase, 0.13 pL Protector RNase inhibitor, and 10.9 pL nuclease free water. Samples are transferred to 200 pL thin-walled PCR tubes and incubated at 50°C for 10 min for reverse transcription. After RT, cells are transferred back to the V-bottom plate. 175 pL of 0.001% Triton-XlOO in PBS-RI is added and cells are centrifuged at 400xg for 7 min at room temperature. Supernatant is removed and the plate is washed once more in 200 pL 0.001% Triton-XlOO in PBS-RI and centrifuged at 400xg for 7 min at room temperature.

DSP cross-linking. Cell pellets are resuspended in 200 pL of 0.25mg/mL Dithiobis (succinimidyl propionate) (DSP) (ThermoFisher, Cat. No. 22585) in IX PBS with 0.2U/pL of Protector RNase Inhibitor for 30 min at room temperature in the dark. 5 pL of IM Tris (pH 7.5) is added to each well to quench reactions at a final concentration of 20 mM Tris for 10 min. The plate is then centrifuged at 400xg for 7 min at room temperature and supernatant is discarded. Cells are washed twice with 200 pL of 0.001% Triton-XlOO in PBS-RI and centrifuged for 10 min, discarding supernatant each time. Remaining cell pellets are resuspended in 50 pL of PBS-RI each. All cells are then transferred into 1 tube for FACS analysis.

Cell-sorting and cDNA extraction. Sorting is performed on a FACSAria™ Fusion (4- way sort) into IgG+ and IgG- populations. The IgG+ population includes cells expressing antigens recognized by patient IgG. All tubes are centrifuged at 400xg for 10 min at room temperature. Supernatant is removed from each tube such that lOOpL is remaining, to preserve the cell pellet. All samples are incubated in 50mM of DL-Dithiothreitol (DTT) (Sigma- Aldrich, Cat. No. D9163) (5.25 pL of IM DTT to lOOpL of sample) for 10 min at room temperature to reverse the DSP crosslinks. 1 OOpL of DNA/RNA Lysis Buffer is added to each tube containing 100 pL of sample (1 : 1 ratio of lysis buffer to sample) and purification proceeds as per gwzcAr-DNA/RNA Microprep Plus kit instructions (Zymo, Cat. No. D7005). Samples are eluted in 22pL of nuclease free water and stored at -20°C until PCR amplification.

Amplification and Sequencing. 8 pL of cDNA from each sorted population is amplified via 20 cycles of PCR using the KAPA2G Fast Multiplex Kit (Roche, Cat. No. 07961430001) and the antigen cDNA primer set. PCR conditions are as follows: 1) 95°C for 3 min, 2) 95°C for 15 sec, 47°C for 30 sec, and 72°C for 30 sec for 20 cycles, 3) 72°C for 1 min. A second PCR is performed to add cell subpopulation-barcoded forward and reverse primers using 2 pL of PCR1 product and primers at 0.25 uM each with the Herculase Fusion DNA Polymerase kit (Agilent. Cat. No. 600677). PCR conditions are as follows: 1) 95°C for 2 min, 2) 95°C for 20 sec, 58°C for 20 sec, and 72°C for 30 sec for 20 cycles, 3) 72°C for 3 min. Barcoded samples are then mixed together and an equal volume of sparQ PureMag beads (Quantabio, Cat. No. 95196-005) are added. Bead clean-up proceeded as per manufacturer’s instructions. Briefly, samples and beads are mixed together until homogenous, incubated at room temperature for 5 min then placed on a magnet. Supernatant is removed and samples are washed twice with 80% ethanol (30 seconds each time) while still on the magnet. Beads are then dried and eluted in nuclease free water. The pooled sample is assessed on a 1% low melting point agarose (Invitrogen, Cat. No. 16520) gel in IX TAE buffer and then gel extracted per Monarch Gel Extraction kit protocol (NEB, Cat. No. T1020S). Sequencing is performed on an Illumina NextSeq with 150bp single-end reads.

Software. Illumina Fastq files are demultiplexed based on sample-specific and sorted population-specific barcodes using the cutAdapt⁵² software with settings allowing for 1 mismatched nucleotide in the barcode. All further data processing is performed using R and Microsoft Excel.

Discussion

Overall, it is expected that INCERTS enables sample mixing for efficient cell sorting and antibody-antigen interaction analysis. Quick, inexpensive INCERTS-based quantification of specific antigen sequences across numerous patient samples and/or over multiple timepoints may also be used to track specific immune responses.

Claims

That Which Is Claimed:

1 . A high throughput method for simultaneously identifying ribonucleic acids (RNA) sequences of interest in a plurality of cell samples comprising the steps of:

(a) fixing the cells present in the cell samples;

(b) permeabilizing the cells;

(c) contacting the permeabilized cells with a plurality of reverse transcription (RT) primers specific for the RNA sequences of interest under conditions that facilitate binding of RT primers to corresponding messenger RNA (mRNA) present in the cells, wherein each RT primer comprises a sample-specific barcode;

(d) reverse transcribing the mRNA to form complementary deoxyribonucleic acid (cDNA) from the mRNA;

(e) mixing the cell samples and sorting into cell subpopulations of interest;

(f) extracting cDNA from at least one of the sorted cell subpopulations; and

(g) sequencing the cDNA, wherein each sequenced cDNA comprises the RNA sequences of interest and sample-specific barcodes from step (c).

2. The method of claim 1 , wherein prior to step (a), after any of steps (a)-(d) or as part of step (e), the method further comprises staining the cells with labeled antibodies that specifically bind cellular components.

3. The method of claims 1 or 2, wherein the sorting of step (e) comprises fluorescence- activated cell sorting (FACS) or magnetic separation.

4. The method of claim 1, wherein after step (f), the method further comprises amplifying the cDNA using primers comprising a cell subpopulation-specific barcode, wherein each sequenced cDNA of step (g) further comprises cell subpopulation-specific barcodes.

5. The method of claim 1 or 4, further comprising the step of deconvoluting the identified RNA sequences of interest back to cell subpopulation and the sample using the sample-specific barcodes and cell subpopulation-specific barcodes.

6. The method of claim 1 or 5, further comprising the step of using complementary patient-matched single-cell RNA-sequencing data to recover full-length sequences for the RNA sequences of interest.

7. The method of claim 1, wherein the cell samples comprise peripheral blood mononuclear cells (PBMCs).

8. The method of claim 1, wherein steps (a) and (b) are performed using a single agent.

9. The method of claim 8, wherein the single agent is methanol.

10. The method of claim 1, wherein after step (d) the method further comprises crosslinking the cells to facilitate intracellular retention of cDNA during cell sorting.

11. The method of claim 10, wherein cross-linking is performed using dithiobis(succinimidyl propionate) (DSP).

12. The method of claim 1, wherein prior to step (a), after any of steps (a)-(d) or as part of step (e), the method further comprises staining the cells with a ligand for a cell surface receptor.

13. The method of claim 12. wherein the ligand comprises antigen for B cells or MHC- peptide multimers for T cells.

14. The method of claim 1 , wherein the RNA sequences of interest comprise T cell receptor (TCR) sequences.

15. The method of claim 14, wherein the cell samples are obtained from patients who have received a candidate peptide vaccine and the method is used to identify reactive TCRs.

16. The method of claim 1, wherein the RNA sequences of interest comprise B cell receptor (BCR) sequences.

17. The method of claim 1, wherein step (d) is performed after step (e).

18. A method for spatial analysis of RNA sequences of interest in a fixed tissue section comprising the steps of:

(a) permeabilizing the cells present in the fixed tissue section;

(b) contacting the permeabilized cells with a plurality of RT primers specific for the RNA sequences of interest under conditions that facilitate binding of RT primers to corresponding mRNA present in the cells, wherein the plurality of RT primers comprises a plurality of barcodes;

(c) reverse transcribing the mRNA to form cDNA;

(d) determining the barcodes in situ, wherein the barcodes represent a spatial location within the cell and tissue section;

(e) sequencing ex situ the cDNA comprising the RNA sequences of interest and spatial barcodes; and

(f) spatially mapping the RNA sequences of interest determined in step (e) using the spatial barcodes determined in step (d).

19. The method of claim 18. wherein step (d) comprises performing in situ sequencing.

20. The method of claim 18, wherein step (d) comprises probe-hybridization analysis.

21. The method of claim 20. wherein the probe-hybridization analysis comprises ligation in situ hybridization Lock’n’Roll.

22. The method of claim 20, wherein the probe hybridization analysis comprises the use of branched chain hybridization.

23. The method of claim 20, wherein the probe hybridization analysis comprises the use of hybridization chain reaction.

24. The method of claim 18. wherein step (d) comprises imaging the spatial barcodes.

25. The method of claim 18, wherein step (d) comprises mass spectrometry' imaging analysis.

26. The method of claim 18, wherein steps (c) and (d) are reversed.

27. The method of claim 18, wherein the RNA sequences of interest comprise TCR sequences, BCR sequences, cancer-associated mutations in RNA, or sequences associated with overexpressed or knocked down genes.

28. A method for detecting antibodies in a plurality of samples comprising the steps of

(a) contacting at least one sample comprising the antibodies with a plurality of cells expressing a plurality of antigens;

(b) fixing the cells;

(c) permeabilizing the cells;

(d) contacting the permeabilized cells with a plurality of RT primers under conditions that facilitate binding of RT primers to antigen-encoding mRNA present in the cells, wherein each RT primer comprises a sample-specific barcode;

(e) reverse transcribing the mRNA to form cDNA;

(f) mixing the cell samples and sorting into cell subpopulations of interest based on the presence of antibodies in the samples;

(g) extracting cDNA from at least one of the sorted cell subpopulations; and

(h) sequencing the cDNA, wherein each sequenced cDNA comprises the antigenencoding mRNA, sample-specific barcodes from step (d) to determine the antigen targets recognized by the antibodies in the samples.

29. The method of claim 28, wherein step (a) is performed after any of steps (b)-(e).

30. The method of claim 28, wherein the sorting of step (f) comprises FACS or magnetic separation.

31. The method of claim 28, further comprising the step of deconvoluting the identified antibody targets via the sample-specific barcodes.

32. The method of claim 28, w herein the sample comprising antibodies comprises serum, plasma, saliva, or cerebrospinal fluid obtained from a patient.

33. The method of claim 28, wherein steps (b) and (c) are performed using a single agent.

34. The method of claim 33, wherein the single agent is methanol.

35. The method of claim 28, wherein prior to step (f), the method further comprises crosslinking the cells to facilitate intracellular retention of cDNA during cell sorting.

36. The method of claim 35, wherein the cross-linking step is performed using dithiobis(succinimidyl propionate) (DSP).

37. The method of claim 28, wherein the plurality of cells overexpresses a library of antigens.

38. The method of claim 28, wherein the subpopulations of step (f) are defined by the presence or absence of specific antibody isotypes.

39. The method of claim 28, wherein the antibodies in the sample comprise viral antibodies and the plurality of cells express a plurality of viral antigens.

40. The method of claim 28, wherein the plurality of cells are human, animal, bacterial or yeast.

41 . The method of claim 28, wherein after step (g), the method further comprises amplify ing the cDNA.

42. The method of claim 41, wherein the amplifying step utilizes primers comprising cell subpopulation-specific barcodes.

43. The method of claim 42, further comprising the step of deconvoluting the identified antibody targets via the sample-specific barcodes and the cell subpopulation-specific barcodes.

44. The method of claim 28, wherein cell subpopulation-specific barcodes are added any time after or as part of step (e) but before step (h).

45. A method for detecting antibodies in a plurality of samples comprising the steps of: (a) contacting at least one sample comprising the antibodies with a plurality of cells expressing a plurality of antigens;

(b) fixing the cells;

(c) permeabilizing the cells;

(d) contacting the permeabilized cells with a plurality of RT primers under conditions that facilitate binding of RT primers to antigen-specific barcodes present in the cells, wherein each RT primer comprises a sample-specific barcode;

(e) reverse transcribing the mRNA to form cDNA;

(f) mixing the cell samples and sorting into cell subpopulations of interest based on the presence of antibodies in the samples;

(g) extracting cDNA from at least one of the sorted cell subpopulations; and

(h) sequencing the cDNA, wherein each sequenced cDNA comprises the antigenspecific barcode, sample-specific barcodes from step (d) to determine the antigen targets recognized by the antibodies in the samples.

46. The method of claim 45. wherein step (a) is performed after any of steps (b)-(e).

47. The method of claim 45, wherein the sorting of step (f) comprises FACS or magnetic separation.

48. The method of claim 45, further comprising the step of deconvoluting the identified antibody targets via the sample-specific barcodes and antigen-specific barcodes.

49. The method of claim 45, wherein the sample comprising antibodies comprises serum, plasma, saliva, or cerebrospinal fluid obtained from a patient.

50. The method of claim 45, wherein steps (b) and (c) are performed using a single agent.

51. The method of claim 50. wherein the single agent is methanol.

52. The method of claim 45, wherein prior to step (f), the method further comprises crosslinking the cells to facilitate intracellular retention of cDNA during cell sorting.

53. The method of claim 52, wherein the cross-linking step is performed using dithiobis(succinimidyl propionate) (DSP).

54. The method of claim 45, wherein the plurality of cells overexpresses a library of antigens.

55. The method of claim 45. wherein the subpopulations of step (f) are defined by the presence or absence of specific antibody isotypes.

56. The method of claim 45, wherein the antibodies in the sample comprise viral antibodies and the plurality of cells express a plurality of viral antigens.

57. The method of claim 45, wherein the plurality of cells are human, animal, bacterial or yeast.

58. The method of claim 45. wherein after step (g), the method further comprises amplify ing the cDNA.

59. The method of claim 58, wherein the amplifying step utilizes primers comprising cell subpopulation-specific barcodes.

60. The method of claim 59, further comprising the step of deconvoluting the identified antibody targets via the sample-specific barcodes, antigen-specific barcodes and the cell subpopulation-specific barcodes.

61. The method of claim 45, wherein the antigen-specific barcodes are encoded on the same mRN A as the antigen.

62. The method of claim 45. wherein cell subpopulation-specific barcodes are added any time after or as part of step (e) but before step (h).