Methods for single-cell RNA and protein sequencing

Abstract

Disclosed herein are methods for preparing a biological sample for single-cell analysis which will simultaneously provide single-cell RNA, single-cell extracellular protein, single-cell cytoplasmic protein, and single-cell transcription factor readouts while preserving cellular heterogeneity. Some methods disclosed herein allow for the detection of proteins including the detection of the level of proteins and / or the detection of post translational modifications of proteins.

Description

METHODS FOR SINGLE-CELL RNA AND PROTEIN SEQUENCINGCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority from U.S. Provisional Application No. 63 / 534,640, filed August 25, 2023, the entire contents of which are incorporated herein by reference.BACKGROUND

[0002] Single-cell transcriptomic and proteomic analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. However, methods of single-cell transcriptomics are unable to simultaneously capture proteins from the same cell and vice versa. Recent studies have indicated that cells fixed by denaturing fixative can be used in single-cell sequencing, however these methods have not been able to capture extracellular and intracellular gene and protein expression simultaneously. The challenge with capturing both intracellular proteins and RNAs is the balance between protein capture and RNA preservation. RNA preservation is difficult as RNA preservation decreases due to degradation, leaking out of the cell, lack of availability due to crosslinking under conditions that enable protein capture.SUMMARY

[0003] Disclosed herein are methods for preparing a biological sample for single-cell analysis which simultaneously provide single-cell RNA, single-cell extracellular protein, single-cell cytoplasmic protein, single-cell transcription factor, and single-cell post-translational modification (PTM) readouts all while preserving cellular heterogeneity.

[0004] In one aspect, the present disclosure is directed to a method of single-cell analysis of total cellular ribonucleic acids (RNAs) and one or more proteins, the method comprising:(a) incubating cells present in single-cell form in methanol;(b) incubating the methanol treated cells in a blocking buffer;(c) incubating the methanol treated cells with an antibody reagent; and(d) detecting total cellular RNAs and one or more proteins in the cells.

[0005] In some embodiments, the cells are incubated in methanol for at least 10 minutes. In some embodiments, the cells are incubated in methanol for at least 30 minutes. In someembodiments, the cells are incubated in methanol at least overnight. Tn some embodiments, the cells are incubated in methanol at a temperature ranging from -100°C to room temperature. In some embodiments, the cells are incubated in methanol at -20°C. In some embodiments, the cells are incubated in methanol at 4°C. In some embodiments, the cells are incubated in methanol at -80°C. In some embodiments, the methanol is a molecular grade methanol that is at least 50% methanol, at least 70% methanol; at least 80% methanol, at least 90% methanol, at least 95% methanol, or 100% methanol.

[0006] In some embodiments, the method further comprises removing methanol after step (a) and resuspending the methanol treated cells in the blocking buffer; then incubating as in step (b).

[0007] In some embodiments, steps (b) and (c) are performed simultaneously.

[0008] In some embodiments, the method comprises step (b) incubating the methanol treated cells in a blocking buffer wherein step (c) is performed after step (b). In some embodiments, the method further comprises, after step (b), adding a wash buffer to methanol treated cells and filtering cells through a sterile filter.

[0009] In some embodiments, the methanol treated cells are incubated in the blocking buffer for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, overnight, 24 hours, or between 10 and 60 minutes.

[0010] In some embodiments, the blocking buffer comprises a saline sodium citrate (SSC) buffer, a DNA blocking agent, a reducing agent, and a RNase inhibitor. In some embodiments, the blocking buffer comprises about 0.1 to 30X SSC buffer, about 0.01% to 5% of Bovine Serum Albumin (BSA), about 0.01 pg / mL to 5000 pg / mL of the DNA blocking agent, about 0.1% to 20% of the reducing agent, about 0.01% to 15% of the RNase inhibitor, and molecular grade / nuclease free water.

[0011] In some embodiments, the SSC buffer comprises sodium chloride, sodium citrate, and / or trisodium citrate. In some embodiments, a formulation of IX SSC comprises a range of about 25 mM to 300 mM NaCl and a range of about 1 mM to 50 mM sodium citrate in a pH range of about 6.5 -7.5. In some embodiments, the IX SSC buffer comprises about 150 mM NaCl and about 15 mM sodium citrate at a pH of about 7.0. In some embodiments, the SSCbuffer is used at a 3X formulation. Tn some embodiments, the SSC buffer is used at a 5X formulation.

[0012] In some embodiments, the method comprises, after incubating the methanol fixed cells in the blocking buffer, removing the blocking buffer; resuspending the methanol treated cells in the antibody reagent; and incubating the methanol treated cells in the antibody reagent.

[0013] In some embodiments, the methanol treated cells are incubated in the antibody reagent at 0°C to room temperature. In some embodiments, the methanol fixed cells are incubated in the antibody reagent for at least 5 minutes. In some embodiments, the methanol fixed cells are incubated in the antibody reagent for at least 16 hours.

[0014] In some embodiments, the antibody reagent comprises 0. IX to 20X Phosphate Buffered Saline (PBS), 0.01 pg / mL to 5000 pg / mL DNA blocking agent, 0.01% to 20% bovine serum albumin, reducing agent, nuclease free water, at least one antibody, and / or an RNase inhibitor.

[0015] In some embodiments, the antibody reagent comprises about 2-8% 20X PBS, about 50 pg / mL to 200 pg / mL of the DNA blocking agent, about 0.4-5% bovine serum albumin, about 1- 4% of the RNase inhibitor, about 0.001 pg / mL to 100 pg / mL of at least one antibody; and / or nuclease free water.

[0016] In some embodiments, the antibody reagent comprises about 4-5% 20X PBS, about 100 pg / mL to 150 pg / mL of the DNA blocking agent, about 0.8-1.2% bovine serum albumin, about 2-3% of the RNase inhibitor, about 0.025 pg / ml to 5pg / ml of at least one antibody, and nuclease free water.

[0017] In some embodiments, the at least one antibody comprises more than one antibody. In some embodiments, the at least one antibody is a conjugated antibody. In some embodiments, the conjugated antibody is conjugated to a DNA sequence or RNA sequence. In some embodiments, the DNA is single stranded DNA. In some embodiments, the DNA is at least 10 nucleotides long. In some embodiments, the DNA ranges between 20 to 100 nucleotides in length.

[0018] In some embodiments, the at least one antibody is an antibody to an individual target protein, a protein complex, a post-translational modification in a protein, and / or a protein / nucleic acid complex. In some embodiments, the target proteins include peptides, enzymes, hormones,and structural components. In some embodiments, the at least one antibody is an antibody directed to: T-bet / TBX21, CD3 (UCHT1), CD8a (SKI), S100A9, CD4 (RPA-T4), TCF1 / TCF7, Ikaros, Aiolos, CD19, Ibal / AIF-1, Phospho-CREB, CREB, Phospho-Akt, Akt, phospho-p44 / 42 MAPK (Erkl / 2), p44 / 42 MAPK (Erkl / 2), Vimentin, phospho-stat3, stat3, phospho-stat4, stat4, phospho-statl, statl, NCAM1, Phospho-Histone H3, histone H3, acetyl-hi stone H3, tri-methyl- histone H3, GAPDH, Mouse (G3A1) mAb IgGl Isotype Control, Phospho-S6 Ribosomal Protein, S6 ribosomal protein, phospho-4E-BPl, CD68, CDl lb / ITGAM, IFI16, phosphor-Zap- 70, Zap-70, NF ATI, BATF, phospho-glucocorticoid receptor, glucocorticoid receptor, Phospho- PLCyl, PLCyl, Phospho-IRF-3, IRF-3, NF-KB p65, phospho-NF-KB p65, FoxOl, PLCyl, phospho-cJun, eJun, IL-17F, IL-17A, caspase-3, phospho-histone H2A.X, histone H2A.X, phospho- YAP, YAP, phospho-p38 MAPK, p38 MAPK, phospho-Rb, Rb (4H1), NRF2, phospho-PTEN, PTEN, PCNA, SMAD2 / 3, phospho- EGF receptor, EGF receptor, c-Myc / N- Myc, phospho-SMAD2, ATF-4, phospho-mTOR, mTOR, phospho-eIF2a, phospho-TBKl / NAK, eIF2a, TBK1 / NAK, Phospho- SAPK / JNK, SAPK / JNK, COL1A1, phospho-c-Fos, c-Fos, Phospho-AMPKa, AMPKa, phospho-Stat6, Stat6, or E-cadherin.

[0019] In some embodiments, the at least one antibody is directed to a post-translational modification of a target protein. In some embodiments, the post-translational modification is phosphorylation, methionine oxidation, deamidation, glycosylation, ubiquitination, carb amyl ati on, S-carboxymethylation, acetylation, or methylation

[0020] In some embodiments, the method further comprises washing the sample with a wash buffer after step (b) and before step (c). In some embodiments, the wash step after step (b) and before step (c) comprises washing the cells with a buffer comprising at least 0.5-10X PBS and / or 0.01-10% BSA; and the wash is optionally repeated at least once. In some embodiments, the wash buffer is a modified wash buffer comprising lx PBS with 0.5% BSA; and optionally RNase inhibitor.

[0021] In some embodiments, the method further comprises washing the sample with a wash buffer after step (c) and before step (d). In some embodiments, the washing step after step (c) and before step (d) is repeated at least once.

[0022] In some embodiments, the method further comprises counting the methanol treated cells.

[0023] In some embodiments, the DNA blocking agent is free DNA from at least one source, free RNA from at least one source, a combination of free DNA from at least two sources, a combination of free RNA from at least two different sources, or a combination of free DNA from at least one source and free RNA from at least one source. In some embodiments, the at least one source for the free DNA is salmon sperm.

[0024] In some embodiments, the total RNAs in the cells are sequenced.

[0025] In some embodiments, the total protein from the methanol treated cells is sequenced from the DNA sequence conjugated on the at least one antibody.

[0026] In some embodiments, the methanol treated cells present in single-cell form are captured using a high throughput single-cell method. In some embodiments, the high throughput method utilizes microfluidics.

[0027] In some embodiments, the method further comprises after step (c), subjecting the cells to single-cell sequencing analysis.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the office upon request and payment of the necessary fee.

[0029] FIG. 1A-C. FIG. 1A-C is a representation showing a comparison and challenges of simultaneous RNA and protein capture for sequencing. FIG. 1 A shows a traditional proteomics capture (CITE-seq) where only extracellular proteins are captured. Additionally, in order to analyze both RNA and protein, three single-cell libraries are used, bridged, and computationally merged. FIG. IB shows the challenges of intracellular protein capture and RNA capture where RNA is degraded and / or lost. FIG. 1C shows a representation of the disclosed method capturing both RNA, extracellular and intracellular proteins as well as transcription factors all using the same single-cell library.

[0030] FIG. 2A-N shows representative readout of single-cell proteomics analysis prepared by the disclosed method. These heat maps show correlation between post-translational modifications of proteins / protein and RNA and other proteins in an unbiased manner. Looking at a target of interest (protein and RNA) in a heterogenous population which aids in finding newtargets for potential therapies in certain diseases relating to cancer, neurodegeneration, and others. FIG. 2A shows a phospho-Stat3 readout. FIG. 2B shows an acetyl-H3 readout. FIG. 2C shows a phospho-ERK readout. FIG. 2D shows a phospho-p38MAPK readout. FIG. 2E shows a phospho-S6 readout. FIG. 2F shows a phospho-H3 readout. FIG. 2G shows a K27me3 readout. FIG. 2H shows a Vimentin readout. FIG. 21 shows Aiolos readout. FIG. 2J shows a LEF1 readout. FIG. 2K shows an IRF4 readout. FIG. 2L shows Ibal-AIFl readout. FIG. 2M shows S100A9 readout. FIG. 2N shows Ikaros readout.

[0031] FIG. 3A-C are representative UMAPs which depict how the disclosed methods do not impact the cellular heterogeneity of the sample. Live peripheral blood mononuclear cells (PBMC) were processed using the disclosed methods. FIG. 3B is the live control sample whereas FIG. 3C is the UMAP representing the cells processed through the disclosed method. The percent cell distribution was also very similar in both control and disclosed methodology conditions, showing that the disclosed methods preserve the heterogeneity in PBMCs.

[0032] FIG. 4A-U are FeaturePlots displaying RNA expression of a target or protein expression of the same target. This assesses the quality of the data using the disclosed methodologies. FIG. 4A shows the clusters of cells and indicates the types of cells in those clusters. FIG. 4B and 4C show the RNA and protein levels of GAPDH, respectively. In FIG. 4P, Ibal RNA is expressed in a cluster of cells while protein expression of Ibal is also seen in the same cluster of cells (4Q). The disclosed methods offer a more accurate representation of the target expression at the protein level as can be seen in that the protein expression is more uniform than the RNA expression, showing one of the benefits of the disclosed methods. The inclusion of FIG. 4D-4U shows how the disclosed technology can display very robust RNA and protein signature, especially when correlated in a single cell dataset.

[0033] FIG. 5A-N is directed to cell type analysis where the disclosed methods are able to facilitate the identification of cell states that are difficult to do through RNA analysis alone. FIG. 5A shows selected CD4 positive T cells from PBMCs, where the data is reanalyzed into three clusters labeled as “naive” CD4 T cells, “effector” CD4 cells, and “memory-like” CD4 cells. FIG. 5B, 5D, and 5F show mapped targeted RNA for three different targets, Stat 3 and TCF7 RNA, while FIG. 6C, 6E, and 6G show mapped targeted protein for three different protein targets, phosphorylated STAT3-Ser705, pSTAT3-Ser727, and TCF1 / TCF7. FIG. 5H showsselected CD8 positive T cells from PBMCs, where the data is reanalyzed into three clusters labeled as “naive” CD8 T cells, “effector” CD8 cells, and “memory-like” CD8 cells. FIG. 51, 5K, and 5M show mapped targeted RNA for three different targets, Stat 3, TCF7, and TBX21 RNA, while FIG. 5 J, 5L, and 5N show mapped targeted protein for three different protein targets, phosphorylated STAT3-Ser727, TCF1 / TCF7, and TBX21. TCF7 is seen to be highly expressed in the naive state of T cells which corroborates with previously published papers. On the other hand, TBX21 was upregulated in the effector / differentiated state. FIG. 5 is a great example showing that analyzing the RNA level for these targets does not show a clear picture of the different cellular states, showing how the intracellular protein and post translational modification readout from an experiment through the disclosed methods offers a new perspective when identifying cellular states.

[0034] FIG. 6A-E shows accurate measurements of post-translational modifications through signaling pathway antibodies. FIG. 6A is a UMAP showing the cells that are control and those that were treated with Wortmannin and LY294002, two highly selective and well-known phosphatidylinositol 3 kinase (PI3K) inhibitors. FIG. 6B shows the level of Akt protein, a protein known to be downstream of PI3K in the signaling pathway, whereas FIG. 6C shows the level of Akt protein phosphorylated at Ser473. As expected, phosphorylation of the downstream Akt protein is inhibited in the cells where PI3K signaling is inhibited. Likewise, FIG. 6D shows the level of S6 protein, a signaling protein known to be downstream of Akt in the PI3K pathway, whereas FIG. 6E shows that the level of phosphorylated S6 is inhibited in the cells treated with PI3K inhibitors.

[0035] FIG. 7A-B represents FeaturePlots of primary T cells that were stimulated with PMA and lonomycin to promote phosphorylation levels since they are generally low in primary cells.

[0036] FIG. 8A-P show FeaturePlots of T cells stimulated with lipopolysaccharide as well as T cells stimulated with lipopolysaccharide and then treated with phosphatase. FIG. 8G shows phospho-Statl (phosphorylated at Serine 727) and total Statl protein and indicates that phospho- Statl (Ser 727) levels were high in stimulated primary T cells (left panel) whereas phosphatase treated cells had very low levels of Phopho-Stat 1 (right panel). A similar pattern with Phospho- Creb (Ser 133) was seen with a decrease in phosphorylation levels after Phosphatase treatment (FIG. 9M). As is seen throughout FIG. 8A-P, total protein levels were not impacted byphosphatase treatment as a control. This data shows that post-translational modifications can also be quantified in primary cells in addition to cell lines.DETAILED DESCRIPTION

[0037] Disclosed herein are methods for preparing a biological sample for single-cell analysis which will simultaneously provide single-cell RNA, single-cell extracellular protein, single-cell cytoplasmic protein, single-cell transcription factor, and post-translational modification (PTM) readouts all while preserving cellular heterogeneity and maintaining a detectable level of RNA in the cells.

[0038] Current single cell RNA-sequencing is a powerful technique for studying the heterogeneity of cells; however, it does not provide any information about protein expression which is essential for understanding the functional state of cells.

[0039] The technology disclosed herein is designed to revolutionize single-cell biology with an efficient and simple experimental workflow. The disclosed methods enable the concurrent measurement of RNA, surface markers, cytoplasmic proteins, and nuclear proteins, within individual cells while also allowing the study of signaling pathways at the single-cell resolution. The methods disclosed herein allow for a deeper understanding of multiple conditions from heterogenous and complex samples. The methods disclosed herein allow the measurement of surface, intracellular, and nuclear proteins, as well as post-translational modifications of those proteins, at the same time as quantifying RNA in single cells. The disclosed methods are also a great screening tool to understand which signaling pathway and RNA targets are impacted in different cell types. Such knowledge will help guide the research and narrow targets as to what the next targets will be. The disclosed methods can also be employed as a great hypothesis generation tool when trying to understand the molecular mechanism in different biological conditions.

[0040] Although claimed subject matter will be described in terms of certain examples, other examples, including examples that do not provide all the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, and process step changes may be made without departing from the scope of the disclosure.

[0041] Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value, the upperlimit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value).

[0042] In the description that follows, certain conventions will be followed as regards to the usage of terminology. Generally, terms used herein are intended to be interpreted consistently with the meaning of those terms as they are known to those of skill in the art. In practicing the present disclosure, many conventional techniques in molecular biology, microbiology, cell biology, biochemistry, and immunology are used, which are within the skill of the art. These techniques are described in greater detail in, for example, Molecular Cloning: a Laboratory Manual 4th edition, J.F. Sambrook and D.W. Russell, ed. Cold Spring Harbor Laboratory Press 2012; Recombinant Antibodies for Immunotherapy, Melvyn Little, ed. Cambridge University Press 2009; “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001). The contents of these references and other references containing standard protocols, widely known to and relied upon by those of skill in the art, including manufacturers’ instructions are hereby incorporated by reference as part of the disclosure.

[0043] Temperatures as used herein are not meant to be exact. -20°C as used herein refers to an ordinary laboratory freezer. -80°C as used herein refers to laboratory deep freezer. 4°C as used herein refers to an ordinary laboratory refrigerator. Room temperature as used herein refers to the normal temperature of a laboratory which is usually around 22°C to 27°C. In some embodiments, steps of the methods can be performed at room temperature while the cells in the single-cell form are on ice. On ice temperature ranges between 0°C and room temperature and is commonplace to those of skill in the art. All temperatures used throughout the disclosure can fluctuate and one having skill in the art understands the definition of these temperatures.

[0044] “Complementary” or “substantially complementary” refers to the hybridization or base pairing or the formation of a duplex between nucleotides or nucleic acids, such as, for instance, between the two strands of a double-stranded DNA molecule or between an oligonucleotideprimer and a primer binding site on a single- stranded nucleic acid. Complementary nucleotides are, generally, A and T (or A and U), or C and G. Two single-stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the other strand, usually at least about 90% to about 95% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, and at least 95%), and even at least about 98% to about 100% (e.g., at least 98%, at least 99%, and 100%).

[0045] As used herein, “filter” or “cell strainer” are devices comprising membranes that are used to sterilize fluid passed through them based on the pore size in the membrane. “Filter” and “cell strainer” are used interchangeably in this disclosure. Types and variations of filters are known in the art. These can include pipette filters, cell strainers, syringe filters, and others.

[0046] “Hybridization” refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide. The resulting (usually) doublestranded polynucleotide is a “hybrid” or “duplex.” “Hybridization conditions” will typically include salt concentrations of approximately less than IM, often less than about 500 mM and may be less than about 200 mM. A “hybridization buffer” is a buffered salt solution such as 5% SSPE, or other such buffers known in the art. Hybridization temperatures can be as low as 5° C, but are typically greater than 22° C, and more typically greater than about 30° C., and typically in excess of 37° C. Hybridizations are often performed under stringent conditions, i.e., conditions under which a primer will hybridize to its target subsequence but will not hybridize to the other, non-complementary sequences. Stringent conditions are sequence-dependent and are different in different circumstances. For example, longer fragments may require higher hybridization temperatures for specific hybridization than short fragments. As other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents, and the extent of base mismatching, the combination of parameters is more important than the absolute measure of any one parameter alone. Generally stringent conditions are selected to be about 5°C lower than the melting temperature for the specific sequence at a defined ionic strength and pH. Exemplary stringent conditions include a salt concentration of at least 0.01 M to no more than IM sodium ion concentration (or other salt) at a pH of about 7.0 to about 8.3 and a temperature of at least 25° C. For example, conditions of 5 x SSPE (750 mM NaCl, 50 mM sodium phosphate, 5 mM EDTA atpH 7.4) and a temperature of approximately 30°C. are suitable for allele-specific hybridizations, though a suitable temperature depends on the length and / or GC content of the region hybridized.

[0047] As used herein, the term "nucleic acid" has its general meaning in the art and refers to refers to a coding or non-coding nucleic sequence. Nucleic acids include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) nucleic acids. Examples of nucleic acid thus include but are not limited to DNA, mRNA, tRNA, rRNA, tmRNA, miRNA, piRNA, snoRNA, and snRNA. Nucleic acids thus encompass coding and non-coding region of a genome (i.e. nuclear or mitochondrial).

[0048] “Nucleic acid”, “oligonucleotide”, “oligo” or grammatical equivalents used herein refers generally to at least two nucleotides covalently linked together. A nucleic acid generally will contain phosphodiester bonds, although in some cases nucleic acid analogs may be included that have alternative backbones such as phosphoramidite, phosphorodithioate, or methylphophoroamidite linkages; or peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with bicyclic structures including locked nucleic acids, positive backbones, non-ionic backbones and non-ribose backbones. Modifications of the ribosephosphate backbone may be done to increase the stability of the molecules; for example, PNA:DNA hybrids can exhibit higher stability in some environments.

[0049] The disclosed methods work with any polynucleotide target. Non-limiting examples of polynucleotide targets, for example gene targets and mRNA targets, include CD4, FOXO1, CD45RO, MYC, IL1R2, PRF1, GZMK, LGALS1, IL17F, IL23R, LYNX1, PRDM1, SELL, SMAD4, ICOS, IKZF5, RORC, AHRR, CTLA4, ITGB7, ENTPD1, CCR8, TSHR, TGFB2, IL12A, IL7R, HLA-DMA, CCR5, TIAF1, BCL6, BHLHE40, CXCR4, and CD307c. Other nonlimiting examples of polynucleotide targets include CD3D, GSTP1, TCF7, CD3E, RNB6, RBI, MYB, CD3G, KRT8, CDH1, ERBB3, ERBB2, TCTN1, ESRI, CDKN1A, TFF3, ABCB1, ABCG2, ADAM23, AKT1, APC, AR, ATM, BAD, BCL2, BIRC5, BRCA1, BRCA2, C4A, CA12, CCNA1, CCND1, CCND2, CCNE1, CDH1, CDH13, CDK2, CD326, CDKN1A, CDKN1C, CDKN2A, CSF1, CST6, CTNNB1, CTSD, EGF, EGFR, EMAP-2, ERBB2, ERBB3, ESRI, ESR2, FOXA1, GATA3, GLI1, GPI, GRB7, GSTP1, HIC1, HPRT1, ID1, IGF1, IGF1R, IGFBP3, IL6, JUN, KRT18, KRT19, KRT5, KRT8, LAMP1, MAPK1, MAPK3, MAPK8, MGMT, MKI67, MLH1, MMP2, MMP9, MUC1, MYB, MYC, NME1, NOTCH1, NR3C1,PGR, PLAU, PRDM2, PSMB2, PSMB4, PTEN, PTGS2, PYCARD, RAB7A, RARA, RARB, RASSF1, RBI, REEP5, RNB6, SERPINE1, SFN, SFRP1, SLC39A6, SLIT2, SNAI2, SRC, TBC1D9. TCTN1, TFF3, TGFB1, THBS1, TP73, TWIST1, VEGFA, XBP1, TCF7, ALCAM, CD25, ITGA6, THY1, PROMI, CXCR4, TP53, MSH2, MLH1, MSH6, PMS2, EPCAM, APC, MEN1, RET, VHL, STAT1, STAT3, STAT4, E2F1, E2F2, E2F3, INSR, ACHE, PIK3CA, PIK3CB, and PZK3CD.

[0050] As used herein, “antibody” may include, for example, monoclonal antibodies, polyclonal antibodies, multivalent antibodies, chimeric antibodies, multispecific antibodies, and antibody fragments that exhibit the desired binding specificity. In some embodiments, nanobodies are used to bind target proteins or analytes. Antibodies to specific proteins may be obtained commercially or generated by methods known in the art. For example, antibodies to specific analytes may be prepared using methods generally disclosed by Howard and Kaser (Making and Using Antibodies: a Practical Handbook, CRC Press, 2007). As used herein, “nanobodies” are the smallest antigen-binding fragment which have complete function.

[0051] In some embodiments, the cellular proteins include an individual target protein, a protein complex, a post-translational modification in a protein, and a protein / nucleic acid complex. In some embodiments, the detection of proteins can include the detection of the level of proteins and / or post translational modifications of proteins. In some embodiments, the antibody conjugate can detect certain post-translational modifications of target proteins. In some embodiments, the detecting includes levels of proteins (quantity), identity of and post- translational modification of proteins, including but not limited to, methylation, acetylation, glycosylation, phosphorylation, and ubiquitination. Protein targets include peptides, enzymes, hormones, structural components such as viral capsid proteins, and antibodies. Protein targets may be synthetic or derived from naturally occurring sources. An individual protein is an isolated polypeptide chain. A protein complex includes two or more polypeptide chains.Samples may include proteins with post translational modifications including but not limited to phosphorylation, methionine oxidation, deamidation, glycosylation, ubiquitination, carb amyl ati on, S-carboxymethylation, acetylation, and methylation. Methods of the disclosure work with any target protein. Non-limiting examples of protein targets include but are not limited to: T-bet / TBX21, CD3 (UCHT1), CD8a (SKI), S100A9, CD4 (RPA-T4), TCF1 / TCF7, Ikaros, Aiolos, CD19, Ibal / AIF-1, Phospho-CREB, CREB, Phospho-Akt, Akt, phospho-p44 / 42MAPK (Erkl / 2), p44 / 42 MAPK (Erkl / 2), Vimentin, phospho-stat3, stat3, phospho-stat4, stat4, phospho-statl, statl, NCAM1, Phospho-Histone H3, histone H3, acetyl-hi stone H3, tri-methyl- histone H3, GAPDH, Mouse (G3A1) mAb IgGl Isotype Control, Phospho-S6 Ribosomal Protein, S6 ribosomal protein, phospho-4E-BPl, CD68, CDl lb / ITGAM, IFI16, phosphor-Zap- 70, Zap-70, NF ATI, BATF, phospho-glucocorticoid receptor, glucocorticoid receptor, Phospho- PLCyl, PLCyl, Phospho-IRF-3, IRF-3, NF-KB p65, phospho-NF-KB p65, FoxOl, PLCyl, phospho-cJun, eJun, IL-17F, IL-17A, caspase-3, phospho-histone H2A.X, histone H2A.X, phospho- YAP, YAP, phospho-p38 MAPK, p38 MAPK, phospho-Rb, Rb (4H1), NRF2, phospho-PTEN, PTEN, PCNA, SMAD2 / 3, phospho- EGF receptor, EGF receptor, c-Myc / N- Myc, phospho-SMAD2, ATF-4, phospho-mTOR, mTOR, phospho-eIF2a, phospho-TBKl / NAK, eIF2a, TBK1 / NAK, Phospho- SAPK / JNK, SAPK / JNK, COL 1 Al, phospho-c-Fos, c-Fos, Phospho-AMPKa, AMPKa, phospho-Stat6, Stat6, and E-cadherin.

[0052] The polynucleotide and protein targets can be related to blood and lymph diseases; cancers; the digestive system; ear, nose, and throat; diseases of the eye; female-specific diseases; male-specific diseases; glands and hormones; heart and blood vessels; diseases of the immune system; male-specific diseases; muscle and bone; neonatal diseases; the nervous system; nutritional and metabolic diseases; respiratory diseases; and / or skin and connective tissue.

[0053] “Primer” means an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Primers usually are extended by a DNA polymerase.

[0054] As used herein, an “antibody or fragment” is a monoclonal antibody, a synthetic antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, a multispecific binding construct that can bind two or more targets, a dual specific antibody, a bi-specific antibody or a multi-specific antibody, or an affinity matured antibody, a single antibody chain or an scFv fragment, a diabody, a single chain comprising complementary scFvs (tandem scFvs) or bispecific tandem scFvs, an Fv construct, a disulfide-linked Fv, a Fab construct, a Fab' construct, a F(ab')2 construct, an Fc construct, a monovalent or bivalent construct from which domains non-essential to monoclonal antibodyfunction have been removed, a single-chain molecule containing one VL, one VH antigen-binding domain, and one or two constant “effector” domains optionally connected by linker domains, a univalent antibody lacking a hinge region, a single domain antibody, a dual variable domain immunoglobulin (DVD-Ig) binding protein or a nanobody. Also included in this definition are antibody mimetics such as affibodies, i.e., a class of engineered affinity proteins, generally small (~6.5 kDa) single domain proteins that can be isolated for high affinity and specificity to any given protein target.

[0055] The “linker” refers to any moiety used to attach or associate the ligand to the polymer construct / oligonucleotide sequence portion of the constructs. Thus, in some embodiments, the linker is a covalent bond. In some embodiments, the linker is a non-covalent bond. In another embodiment the linker is composed of at least one to about 25 atoms. Thus, in some embodiments, the linker is formed of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 atoms. In some embodiments, the linker is at least one to about 60 nucleic acids. Thus in some embodiments, the linker is formed of a sequence of at least 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, up to 60 nucleic acids. In some embodiments, the linker refers to at least one to about 30 amino acids. Thus, in some embodiments, the linker is formed of a sequence of at least 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, up to about 30 amino acids. In some embodiments, the linker can be a larger compound or two or more compounds that associate covalently or non-covalently. In some embodiments, the linker can be a combination of the linkers defined herein. The linkers used in the constructs of the compositions and methods are in some embodiments cleavable. The linkers used in the constructs of the methods are in some embodiments non-cleavable. Without limitation, in some embodiments, the linker is a cleavable linker, e.g., disulfide bond or photocleavable bond. In some embodiments, linker comprises a complex of biotin bound to the construct oligonucleotide sequence by a disulfide bond, with streptavidin fused to the ligand. In some embodiments, the biotin is bound to the ligand and the streptavidin is fused to the construct oligonucleotide sequence. In some embodiments, the linker is bound to the 5' end of the oligonucleotide of the construct. In some embodiments, the linker may be covalently attached or conjugated other than covalently to any oligonucleotide sequence portion of the construct. In some embodiments,when the ligand is a recombinant or synthesized antibody, the linker can be engineered into the antibody sequence to facilitate 1 : 1 coupling to the polymer construct, thereby simplifying manufacturing of the ligand, the construct and / or the polymer construct. For example, a Halotag® linker can be engineered into the selected ligand (e.g., antibody) or into the polymer construct or component, for such purposes. Addtionally or alternatively, the ligand is linked to the polymer construct upon production in the same cell. See, e.g., the Halotag® protocols described by Flexi® Vector Systems Technical Manual (TM254 -revised 5 / 17), copyright 2017 by Promega Corporation; and Janssen D. B., “Evolving haloalkaline dehalogenase”, Curr. Opin. Chem. Biol., 2004, 8: 150-159.

[0056] The “polymer construct” or “construct oligonucleotide sequence” is the portion of the construct which is associated with the ligand. This association can be covalent, non-covalent or by any suitable conjugation and employing any suitable linker. The polymer construct is formed by a series of functional polymeric elements, e.g., nucleic acid sequences or other polymers as defined above, each having a function as defined herein. The ligand can be attached to the construct oligonucleotide sequence at its 5' end or at any other portion, provided that the attachment or conjugation does not prevent the functions of the components of the construct oligonucleotide sequence. These components are for each “first” or “additional” construct oligonucleotide sequence, an Amplification Handle; a Barcode, an optional UMI and an Anchor. In general, the polymer construct can be any length that accommodates the lengths of its functional components. In some embodiments, the polymer construct is between 20 and 100 monomeric components, e.g., nucleic acid bases, in length. In some embodiments, the construct oligonucleotide sequence is at least 20, 30, 40, 50, 60, 70, 80, 90 or over 100 monomeric components, e.g., nucleic acid bases, in length. In some embodiments, the construct oligonucleotide is 200 to about 400 monomeric components, e.g., nucleotides, in length. In some embodiments, the polymer construct is generally made up of deoxyribonucleic acids (DNA). In some embodiments, the construct oligonucleotide is a DNA sequence. In some embodiments, the construct oligonucleotide, or portions thereof, comprises modified DNA bases. Modification of DNA bases are known in the art and can include chemically modified bases including labels. In other embodiments, the construct oligonucleotide, or portions thereof, comprises ribonucleic acid (RNA) sequences or modified ribonucleotide bases. Modification of RNA bases are known in the art and can include chemically modified bases including labels. In some embodiments,different portions of the construct oligonucleotide sequence can comprise DNA and RNA, modified bases, or modified polymer connections (including but not limited to PNAs and LNAs). Oligonucleotide modifications are known in the art and customized oligonucleotide modifications are available commercially. In some embodiments, the polymer construct is composed of polyamides, PNA, etc.

[0057] As used herein, the term “Amplification Handle” refers to a functional component of the construct oligonucleotide sequence which itself is an oligonucleotide or polynucleotide sequence that provides an annealing site for amplification of the construct oligonucleotide sequence. The Amplification Handle can be formed of polymers of DNA, RNA, PNA, modified bases or combinations of these bases, or polyamides, etc. In some embodiments, the Amplification Handle is about 10 of such monomeric components, e.g., nucleotide bases, in length. In some embodiments, the Amplification Handle is at least about 5 to 100 monomeric components, e.g., nucleotides, in length. Thus in some embodiments, the Amplification Handle is formed of a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 monomeric components, e.g., nucleic acids. In some embodiments, when present in first or additional construct oligonucleotide sequences, the Amplification Handle can be the same or different, depending upon the techniques intended to be used for amplification. In some embodiments, the Amplification Handle can be a generic sequence suitable as an annealing site for a variety of amplification technologies. Amplification technologies include, but are not limited to, DNA-polymerase based amplification systems, such as polymerase chain reaction (PCR), real-time PCR, loop mediated isothermal amplification (LAMP, MALBAC), strand displacement amplification (SDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA) and polymerization by any number of DNA polymerases (for example, T4 DNA polymerase, Sulfulobus DNA polymerase, Klenow DNA polymerase, Bst polymerase, Phi29 polymerase) and RNA-polymerase based amplification systems (such as T7-, T3-, and SP6-RNA-polymerase amplification), nucleic acid sequence based amplification (NASBA), self-sustained sequence replication (3 SR), rolling circle amplification (RCA), ligasechain reaction (LCR), helicase dependent amplification (HDA), ramification amplification method and RNA-seq.

[0058] The term “Barcode” or “construct Barcode” describes a defined polymer, e.g., a polynucleotide, which when it is a functional element of the polymer construct, is specific for a single ligand. As used in the various methods described herein the term Barcode can be a “cell Barcode” or “substrate Barcode”, which describes a defined polynucleotide, specific for identifying a particular cell or substrate. In some embodiments, the Barcode can be formed of a defined sequence of DNA, RNA, modified bases or combinations of these bases, as well as any other polymer defined above. In some embodiments, the Barcode is about 2 to 4 monomeric components, e.g., nucleotide bases, in length. In other embodiments, the Barcode is at least about 1 to 100 monomeric components, e g., nucleotides, in length. Thus, in some embodiments, the Barcode is formed of a sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94,95, 96, 97, 98, 99 or up to 100 monomeric components, e.g., nucleic acids. In some embodiments the barcode is present along with a Unique Molecular Identifier (UMI). In some embodiments, the barcode is present without a UMI.

[0059] The term “Unique Molecular Identifier” (UMI), also called equivalently a “Random Molecular Tag” (RMT), is a random sequence of monomeric components of a polymer as described above, e.g., nucleotide bases, which when it is a functional element of the polymer construct, is specific for that polymer construct. The UMI permits identification of amplification duplicates of the polymer construct / construct oligonucleotide sequence with which it is associated. In some embodiments, one or more UMI may be associated with a single polymer construct / construct oligonucleotide sequence. The UMI may be positioned 5' or 3' to the Barcode in the composition. In some embodiments, the UMI may be inserted into the polymer / construct oligonucleotide sequence as part of the described methods. In some embodiments, depending on which RNA-sequencing method is used, a UMI is added during the method. However, not all RNA-seq methods make use of UMIs. For example, in single-cell droplet RNA-sequencing, another UMI is introduced during reverse transcription. Each UMI is specificfor its construct oligonucleotide sequence. Thus, when the compositions or methods comprise multiple “first constructs”, each first construct differs only in the sequence of its UMI. Each additional construct will also have its own UMI, which is not present on duplicate additional constructs or additional constructs that differ from each other in target, ligand, Barcode and Anchor specificity. Similarly, as used in the various methods described herein, a UMI may be associated with a polymer, e.g., an oligo or polynucleotide sequence, used in a particular assay format or with a polymer, e.g., an oligo or polynucleotide, that is immobilized on a substrate. Each UMI for each polymer construct, e g., oligonucleotide or polynucleotide, is different from any other UMI used in the compositions or methods. In some embodiments, the UMI is formed of a random sequence of DNA, RNA, modified bases or combinations of these bases or other monomers of the polymers identified above. In some embodiments, a UMI is about 8 monomeric components, e.g., nucleotides, in length. In some embodiments, each UMI can be at least about 1 to 100 monomeric components, e.g., nucleotides, in length. Thus in some embodiments, the UMI is formed of a random sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,80, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 monomeric components, e.g., nucleic acids. In some embodiments, the UMI is formed by a non-random oligonucleotide sequence.

[0060] As used herein, the term “Anchor” refers to a defined polymer, e.g., a polynucleotide or oligonucleotide sequence, which is designed to hybridize to another oligonucleotide sequence, e.g., a capture polymer, a capture oligonucleotide, a primer and the like. In one embodiment of the polymer construct, an Anchor is designed for the purpose of generating a double-stranded construct oligonucleotide sequence. In some embodiments, the Anchor is positioned at the 3' end of an oligonucleotide sequence (e.g., a construct oligonucleotide sequence). In some embodiments, an Anchor is positioned at the 5' end of a construct oligonucleotide sequence. In some embodiments, each Anchor is specific for its intended complementary sequence. For example, in some embodiments, an anchor is configured to hybridize to a 3' end of a capture oligonucleotide such that the 3' end of the capture oligonucleotide acts as a primer that can generate a second complementary strand of the oligonucleotide in the presence of a polymerase. In some embodiments, when the compositions or methods comprise multiple “first constructs”,each first construct has the same Anchor sequence. Tn some embodiments, each additional Anchor has a different additional sequence which hybridizes to a different complementary sequence. In some embodiments, each additional Anchor may have the same Anchor sequence as the first or other constructs, depending upon the assay method steps. When used in the various methods described herein, an Anchor may hybridize to a free complementary sequence or with a complementary sequence that is immobilized on a substrate. In some embodiments, the Anchor can be formed of a sequence of monomers of the selected polymer, e.g., DNA, RNA, modified bases or combinations of these bases, PNAs, polyamides, etc. In some embodiments, an Anchor is about 3 to 15 monomeric components, e.g., nucleotides, in length. In some embodiments, each Anchor can be at least about 3 to 100 monomeric components, e.g., nucleotides, in length. In some embodiments, an anchor comprises 3 to 100, 3 to 50, 3 to 30, 5 to 30, 10 to 20, 5 to 20, or 5 to 15 monomeric components (e.g., nucleotides in length). In some embodiments, an Anchor is formed of a sequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92,93, 94, 95, 96, 97, 98, 99 or up to 100 monomeric components, e.g., nucleic acids. In some embodiments, an Anchor sequence comprises or consists of a polyA sequence. In some embodiments, a polyA sequence comprises a nucleic acid sequence comprising ten or more (e.g., 10-40, 10-30 or 10-20) consecutive adenosine nucleotides, derivatives or variants of an adenosine nucleotide, the like, or a combination thereof. In some embodiments, an Anchor sequence comprises or consists of a polyT sequence. In some embodiments, an Anchor sequence is a polyG sequence. In some embodiments, an Anchor sequence may be a random sequence provided that it can hybridize to its intended complementary sequence (e.g., a capture oligonucleotide, amplification primer, or the like). For example, in some embodiments a method described herein may utilize a plurality of oligonucleotides (e.g., a plurality of constructs comprising a ligand attached to an oligonucleotide), where some or all of the oligonucleotides comprise a different anchor (i.e., an anchor having a different nucleic acid sequence, or an anchor having a substantially different nucleic acid sequence). Some embodiments of the methods described herein utilize a plurality of oligonucleotides (e.g., a plurality of constructs comprising a ligand attached to an oligonucleotide), where some or all of the oligonucleotidescomprise the same anchor. Some embodiments of methods described herein utilize a plurality of oligonucleotides (e.g., a plurality of constructs comprising a ligand attached to an oligonucleotide), where some or all of the oligonucleotides comprise an anchor that is substantially identical (e.g., comprising a nucleic acid sequence that is substantially identical). In some embodiments, a method described herein utilizes a plurality of oligonucleotides (e.g., a plurality of constructs comprising a ligand attached to an oligonucleotide), where some or all of the oligonucleotides comprise an anchor comprising a polyA sequence. In some embodiments, the polyA sequence of a plurality of anchors is substantially identical. As understood by one of skill in the art, polyA sequences that are substantially identical may differ substantially in length. In some embodiments, a polyA sequence (e.g, a polyA sequence of an anchor) is a nucleic acid configured to hybridize to a polyT sequence (e.g., an oligonucleotide or capture oligonucleotide comprising a polyT sequence). As understood by one of skill in the art, depending on hybridization conditions a polyA sequence may comprise one, two, three or four non-polyA nucleotides and still hybridize efficiently to a polyT sequence, thereby providing an annealed polyA-polyT complex comprising one, two, three or more mismatches. Accordingly, in some embodiments, a polyA sequence is a nucleic acid sequence comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% adenosine nucleotides, adenosine analogs, adenosine variants or a combination thereof.

[0061] In some embodiments, an oligonucleotide comprises a polyT sequence. In some embodiments, a capture oligonucleotide comprises a polyT sequence (e.g., a 3' polyT sequence). In some embodiments a method described herein may utilize a plurality of oligonucleotides (e.g., a plurality of capture oligonucleotides), where some or all of the oligonucleotides comprise a polyT sequence. In some embodiments, a polyT sequence of a plurality of oligonucleotides is substantially identical. In some embodiments, a plurality of capture oligonucleotides (e.g., a plurality of different capture oligonucleotides, e.g., different bead-specific capture oligonucleotides) comprise a polyT sequence that is substantially identical. As understood by one of skill in the art, polyT sequences that are substantially identical may differ substantially in length. In some embodiments, a polyT sequence comprises 3 to 100, 3 to 50, 3 to 30, 5 to 30, 10 to 20, 5 to 20, or 5 to 15 consecutive nucleotides (e.g., nucleotides in length). In certain embodiments, a polyT sequence comprises a nucleic acid sequence comprising three or more, ten or more, 3 to 100, 3 to 50, 3 to 30, 5 to 30, 10 to 20, 5 to 20, or 5 to 15 consecutive thymidinenucleotides, derivatives or variants of a thymidine nucleotide, the like, or a combination thereof In some embodiments, a polyT sequence (e.g, a polyT sequence of a capture oligonucleotide) is a nucleic acid configured to hybridize to a polyA sequence. As understood by one of skill in the art, depending on hybridization conditions, a polyT sequence may comprise one, two, three or four non-thymidine nucleotides and still hybridize efficiently to a polyA sequence, thereby providing an annealed polyA-polyT complex comprising one, two, three or more mismatches. Accordingly, in some embodiments, a polyT sequence is a nucleic acid sequence comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% thymidine nucleotides, thymidine analogs, thymidine variants or a combination thereof. In some embodiments, a polyT sequence comprises one or more uracil nucleotides, or derivative thereof.

[0062] The “capture oligonucleotide” or “capture oligo” or “capture polymer” is a polymeric sequence, e.g., an oligonucleotide, comprising at least a sequence that is complementary to an Anchor. In some embodiments, the capture polymer / oligo is not part of the first or additional constructs; rather it is any polymeric sequence or oligonucleotide belonging to a constructpurification kit or an mRNA-sequencing kit. As used herein, the term “complementary sequence” refers to a sequence to which an Anchor sequence (or other nucleic acid, e.g., a primer or capture oligonucleotide) is intended to hybridize to, often resulting in a hybridized double stranded complex. In the presence of a polymerase, a hybridized complex can often be extended in a 3' direction where a nucleic acid template is present. Accordingly, in some embodiments, a sequence complementary to an anchor can hybridize to an anchor sequence thereby providing a primer for amplification and / or to generate a double stranded sequence. In some embodiments, the capture polymer / oligonucleotide sequence may contain sequences that can be used as Amplification Handles and optionally one or more Unique Molecular Identifiers and Barcode sequences. In the methods disclosed herein, the extension of the capture polymer / oligonucleotide with its complementary sequence hybridized to the Anchor sequence copies the Barcode, the UMI and the Amplification Handle from the first or additional constructs onto the capture polymer / oligonucleotide. In any embodiment, the capture polymer / oligonucleotide and its complementary sequence can be formed of DNA, RNA, modified bases or combinations of these bases or of any other polymeric component as defined above. Depending upon the assay steps involved and the intended target, the capture sequence can be unhindered or “free” in the biological sample. In one embodiment, the capturepolymer / oligo contains a complementary sequence that is a primer sequence designed to participate in amplifying the polymer construct / construct oligonucleotide sequence. In another embodiment, the capture sequence is immobilized on a substrate. Similarly to the Anchor sequence, each capture sequence can be at least about 3 to about 100 monomeric units, e.g., nucleotides, in length. Thus in various embodiments, the capture or its complementary sequence is formed of a sequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 monomeric units, e.g., nucleic acids. In some embodiments, a capture oligo comprises a complementary sequence polyT sequence when the Anchor sequence is a polyA sequence. In some embodiments, the capture oligo contains a polyA sequence. In some embodiments, the complementary sequence may be a random polymer, e.g., oligonucleotide sequence, provided that it can hybridize to its intended Anchor sequence.

[0063] “Sequencing,” “sequence determination” and the like means determination of information relating to the nucleotide base sequence of a nucleic acid. Such information may include the identification or determination of partial as well as full sequence information of the nucleic acid. Sequence information may be determined “with varying degrees of statistical reliability or confidence. In one aspect, the term includes the determination of the identity and ordering of a plurality of contiguous nucleotides in a nucleic acid. “High throughput digital sequencing” or “next generation sequencing” means sequence determination using methods that determine many (typically thousands to billions) of nucleic acid sequences in an intrinsically parallel manner, i.e. where DNA templates are prepared for sequencing not one at a time, but in a bulk process, and where many sequences are read out preferably in parallel, or alternatively using an ultra-high throughput serial process that itself may be parallelized. Such methods include but are not limited to pyrosequencing (for example, as commercialized by 454 Life Sciences, Inc., Branford, Conn.); sequencing by ligation (for example, as commercialized in the SOLiD™ technology, Life Technology, Inc., Carlsbad, Calif.); sequencing by synthesis using modified nucleotides (such as commercialized in TruSeq™ and HiSeg™ technology by Illumina, Inc., San Diego, Calif, HeliScope™ by Helicos Biosciences Corporation, Cambridge, Mass., and PacBio RS by Pacific Biosciences of California, Inc., Menlo Park, Calif), sequencingby ion detection technologies (Ion Torrent, Inc., South San Francisco, Calif.); sequencing of DNA nanoballs (Complete Genomics, Inc., Mountain View, Calif.); nanopore-based sequencing technologies (for example, as developed by Oxford Nanopore Technologies, LTD, Oxford, UK), and like highly parallelized sequencing methods.

[0064] Single-cell assays are part of the present disclosure (see, e.g., Ryan et al., Biomicrofluidics 5, 021501 (2011) for an overview of applications of microfluidics to assay individual cells). A single-cell assay may be contemplated as an experiment that quantifies a function or property of an individual cell when the interactions of that cell with its environment may be controlled precisely or may be isolated from the function or property under examination.

[0065] A "biological sample" may be obtained directly from an organism or from a sample obtained from an organism, e g., from blood, urine, cerebrospinal fluid, seminal fluid, saliva, sputum, stool, and tissue. Any tissue or body fluid specimen may be used as a source for single cells for use in the disclosed methods. The cells used in the disclosure can also be from a biological sample or cultured cells, such as a primary cell culture or a cell line. The cells or tissues from which the cells are obtained may be infected with a virus or other intracellular pathogen. In some embodiments, the sample may be from a mammal. In some embodiments, the sample may be from an animal. The term "animal" includes mammals, for example, human, horse, camel, dog, cat, pig, cow, goat and sheep.General Description

[0066] One aspect of the disclosure is directed to a method of single-cell analysis of total cellular ribonucleic acids (RNAs) and one or more proteins, the method comprising:(a) incubating cells present in single-cell form in methanol;(b) incubating the methanol treated cells in a blocking buffer;(c) incubating the methanol treated cells with an antibody reagent; and(d) detecting total cellular RNAs and one or more proteins in the cells.

[0067] In some embodiments, the cells are collected as samples from mammals. In some embodiments, the cells are collected from humans. In some embodiments, the cells are collected from animals.

[0068] The term “single-cell form” as used herein, means that the cells in a sample are isolated and separated one from another. Methods of dissociation are known in the art and there are numerous commercially available kits for dissociating samples to single cells or isolated nuclei. There are several ways to isolate individual cells prior to amplification and sequencing which are known in the art. Fluorescence-activated cell sorting (FACS) is a widely used approach. Magnetic-activated cell sorting (MACS) uses antibody-mediated superparamagnetic nanoparticles to tag specific proteins on the target cells. However, this means that, unlike with FACS, only cell surface molecules can be used as a target to tag live cells. An external magnetic field is then used to isolate the tagged cells while the others are washed away. Therefore, the purity of a MACS isolation depends on the specificity and affinity of the antibodies used for tagging. Individual cells can also be collected by micromanipulation, for example by serial dilution or by using a patch pipette or nanotube to harvest a single cell. The advantages of micromanipulation are ease and low cost, but they are laborious and susceptible to misidentification of cell types under microscope. Dissociation, including mechanical, enzymatic, or chemical, is another method of collecting single cells. Mechanical dissociation refers to breaking down tissue with physical force, such as cutting, crushing, or scraping. Enzymatic dissociation of tissue uses specific enzymes that digest samples to release the target cells. Nonlimiting examples of enzymes used for dissociation include collagenase, trypsin, papain, and elastase. Chemical dissociation takes advantage of the cations that hold together intracellular bonds by adding a chemical compound with an affinity to cations that causes bonds within the sample to dissolve. EGTA, or egtazic acid, is an example of a compound used in chemical dissociation. Laser-capture microdissection (LCM) can also be used for collecting single cells. Although LCM preserves the knowledge of the spatial location of a sampled cell within a tissue, it is hard to capture a whole single cell without also collecting the materials from neighboring cells. In some embodiments, single nuclei isolation is used in sample preparation where the isolation of single nuclei is performed by reagents such as triton X, tween 20, NP-40, and CHAPS. High-throughput methods for single cell isolation also include utilizing microfluidics or microfluidic device, such as methodologies and devices used in single cell sequencing. Both FACS and microfluidics are accurate, automatic, and capable of isolating unbiased samples. However, both methods require detaching cells from their microenvironments first, thereby causing perturbation to the transcriptional profiles in RNA expression analysis.Step (a) - Incubating cells present in single-cell form in methanol.

[0069] In some embodiments, the cells are incubated in methanol for at least 10 minutes. In some embodiments, the cells are incubated in methanol for at least 20 minutes. In some embodiments, the cells are incubated in methanol for at least 30 minutes. In some embodiments, the cells are incubated in methanol for at least 60 minutes. In some embodiments, the cells are incubated in methanol for at least 2 hours. In some embodiments, the cells are incubated in methanol for at least 4 hours. In some embodiments, the cells are incubated in methanol for at least 8 hours. In some embodiments, the cells are incubated in methanol for at least 12 hours. In some embodiments, the cells are incubated in methanol for at least 16 hours. In some embodiments, the cells are incubated in methanol overnight. As used herein, “overnight” means at least 12 hours but no more than 24 hours. In some embodiments, the cells are incubated in methanol for at least 2 nights. In some embodiments, the cells are incubated in methanol for at least 3 nights. In some embodiments, the cells can be incubated in methanol at -80°C for at least 4 days. In some embodiments, the cells are incubated in methanol at -80°C for at least 7 days. In some embodiments, the cells are incubated in methanol at -80°C for at about 2 weeks to 10 months.

[0070] In some embodiments, the cells are incubated in methanol at a temperature ranging from -100°C to room temperature. In some embodiments, the cells are incubated at -80°C. In some embodiments, the cells are incubated at -20°C. In some embodiments, the cells are incubated at 4°C. In some embodiments, the cells are incubated at room temperature.

[0071] In some embodiments, the cells are incubated in methanol. In some embodiments, the methanol is a molecular grade methanol. In some embodiments, the methanol is at least 50% methanol. In some embodiments, the methanol is at least 60% methanol. In some embodiments, the methanol is at least 70% methanol. In some embodiments, the methanol is at least 80% methanol. In some embodiments, the methanol is at least 90% methanol. In some embodiments, the methanol is at least 95% methanol. In some embodiments, the methanol is at least 96% methanol. In some embodiments, the methanol is at least 97% methanol. In some embodiments, the methanol is at least 98% methanol. In some embodiments, the methanol is at least 99% methanol. In some embodiments, the methanol is 100% methanol.Step (b) - Incubating the Methanol Treated Cells in a Blocking Buffer.

[0072] In some embodiments, the methanol is removed after step (a) and the methanol treated cells are resuspended in the blocking buffer. Then, the methanol treated cells are incubated in the blocking buffer, step (b).

[0073] In some embodiments, the methanol treated cells are washed in a buffer before being incubated in blocking buffer. The buffer used to wash the methanol treated cells can be the blocking buffer, a phosphate buffered saline (PBS), a wash buffer, or other buffers known in the art. In some embodiments, the wash buffer comprises PBS with bovine serum albumin (BSA). In some embodiments, the methanol treated cells are washed more than once.

[0074] In some embodiments, the methanol treated cells are removed from the methanol, resuspended, and filtered through a sterile filter or cell strainer. In some embodiments, the methanol treated cells are removed from the methanol, resuspended, washed at least once, and filtered through a sterile filter. In some embodiments, the sterile filter has a pore size ranging in size from about 10 microns to about 100 microns. In some embodiments, the sterile filter has a pore size ranging in size from about 20 microns to about 90 microns. In some embodiments, the sterile filter has a pore size ranging in size from about 30 microns to about 80 microns. In some embodiments, the sterile filter has a pore size ranging in size from about 40 microns to about 70 microns. In some embodiments, the sterile filter has a pore size of about 10 microns. In some embodiments, the sterile filter has a pore size of about 20 microns. In some embodiments, the sterile filter has a pore size of about 30 microns. In some embodiments, the sterile filter has a pore size of about 40 microns. In some embodiments, the sterile filter has a pore size of about 50 microns.

[0075] In some embodiments, the methanol treated cells are incubated in blocking buffer for at least 5 minutes. In some embodiments, the methanol treated cells are incubated in blocking buffer for at least about 10 minutes. In some embodiments, the methanol treated cells are incubated in blocking buffer for at least about 20 minutes. In some embodiments, the methanol treated cells are incubated in blocking buffer for at least about 30 minutes. In some embodiments, the methanol treated cells are incubated in blocking buffer for at least about 45 minutes. In some embodiments, the methanol treated cells are incubated in blocking buffer for at least about 60 minutes. In some embodiments, the methanol treated cells are incubated inblocking buffer between about 10 and about 60 minutes. Tn some embodiments, the methanol treated cells are incubated in blocking buffer overnight. In some embodiments, the methanol treated cells are incubated in blocking buffer for about 24 hours.

[0076] In some embodiments, the methanol treated cells are incubated in blocking buffer at 4°C. In some embodiments, the methanol treated cells are incubated in blocking buffer at room temperature. In some embodiments, the methanol treated cells are incubated in blocking buffer on ice.Step (C) - Incubating the Methanol Treated Cells with an Antibody Reagent.

[0077] Step (c) of the disclosed method is incubating the methanol treated cells with an antibody reagent.

[0078] In some embodiments, step (b) of incubating the methanol treated cells in a blocking buffer occurs before step (c), the step of incubating the methanol treated cells with an antibody reagent. In some embodiments, step (b), incubating the methanol treated cells in a blocking buffer, and step (c), incubating the methanol treated cells with an antibody reagent, occur simultaneously.

[0079] In some embodiments, after the methanol treated cells have incubated in blocking buffer, the blocking buffer is removed; the methanol treated cells are resuspended in the antibody reagent; and the methanol treated cells are incubated in the antibody reagent.

[0080] In some embodiments, the method comprises washing the methanol treated cells with a buffer after step (b) and before step (c). The buffer used in this wash can be the same buffer as was optionally used between steps (a) and (b) described above. The buffer used to wash the methanol treated cells can be the blocking buffer, a phosphate buffered saline (PBS), a wash buffer, or other buffers known in the art. In some embodiments, the wash buffer comprises PBS with bovine serum albumin (BSA). In some embodiments, the methanol treated cells are washed more than once.

[0081] In some embodiments, the methanol treated cells are removed from the blocking buffer, resuspended, and fdtered through a sterile fdter. In some embodiments, the methanol treated cells are removed from the blocking buffer, resuspended, washed at least once, and filtered through a sterile filter.

[0082] In some embodiments, the methanol treated cells are incubated in the antibody reagent at a range of 0°C to room temperature. In some embodiments, the methanol treated cells are incubated in the antibody reagent at 4°C. In some embodiments, the methanol treated cells are incubated in the antibody reagent at room temperature. In some embodiments, the methanol treated cells are incubated in the antibody reagent on ice.

[0083] In some embodiments, the methanol fixed cells are incubated in the antibody reagent for at least 5 minutes. In some embodiments, the methanol fixed cells are incubated in the antibody reagent for at least 10 minutes. In some embodiments, the methanol fixed cells are incubated in the antibody reagent for at least 15 minutes. In some embodiments, the methanol fixed cells are incubated in the antibody reagent for at least 20 minutes. In some embodiments, the methanol fixed cells are incubated in the antibody reagent for at least 30 minutes. In some embodiments, the methanol fixed cells are incubated in the antibody reagent for at least 60 minutes. In some embodiments, the methanol fixed cells are incubated in the antibody reagent for at least 120 minutes. In some embodiments, the methanol fixed cells are incubated in the antibody reagent for at least 4 hours. In some embodiments, the methanol fixed cells are incubated in the antibody reagent for at least 8 hours. In some embodiments, the methanol fixed cells are incubated in the antibody reagent for at least 12 hours. In some embodiments, the methanol fixed cells are incubated in the antibody reagent for at least 16 hours. In some embodiments, the methanol fixed cells are incubated in the antibody reagent for at least 20 hours. In some embodiments, the methanol fixed cells are incubated in the antibody reagent overnight. In some embodiments, the methanol fixed cells are incubated in the antibody reagent for about 48 hours. It is to be understood that at some time point, the loss of RNA outweighs the benefit of incubation time for protein capture. The balance between time of antibody incubation for solid protein signal and the loss of RNA due to degradation, RNA leaking out of the cell, lack of availability of RNA due to crosslinking can be monitored and will differ depending on the cell type, target proteins, and target RNA.Steps (b) and (c)

[0084] In some embodiments, step (b) and step (c) are performed at the same time, i.e., the methanol treated cells are incubated with a blocking buffer and an antibody reagent at the same time. In some embodiments, step (b) and step (c) are performed sequentially, e.g., the methanoltreated cells are incubated with a blocking buffer, and subsequently, the cells are incubated with an antibody reagent.Step (d) - Detecting Total Cellular RNAs and Proteins in the Cells.

[0085] In some embodiments, after the completion of step (c), the methanol treated cells are prepared for detection of cellular RNA and proteins. In some embodiments, the methanol treated cells are washed with a buffer after step (c) and before step (d).

[0086] The disclosed methods maintain cellular RNA in the samples at detectable levels. In some embodiments, between about 40% and about 100% of the cellular RNA is maintained as compared to a live control. In some embodiments, between about 45% and about 95% of the cellular RNA is maintained as compared to a live control. In some embodiments, between about 50% and about 90% of the cellular RNA is maintained as compared to a live control. In some embodiments, between about 55% and about 85% of the cellular RNA is maintained as compared to a live control. In some embodiments, between about 60% and about 80% of the cellular RNA is maintained as compared to a live control. In some embodiments, between about 60% and about 75% of the cellular RNA is maintained as compared to a live control. In some embodiments, at least about 40% of the cellular RNA is maintained as compared to a live control. In some embodiments, at least about 45% of the cellular RNA is maintained as compared to a live control. In some embodiments, at least about 50% of the cellular RNA is maintained as compared to a live control. In some embodiments, at least about 55% of the cellular RNA is maintained as compared to a live control. In some embodiments, at least about 60% of the cellular RNA is maintained as compared to a live control. In some embodiments, at least about 65% of the cellular RNA is maintained as compared to a live control. In some embodiments, at least about 70% of the cellular RNA is maintained as compared to a live control. In some embodiments, at least about 75% of the cellular RNA is maintained as compared to a live control. In some embodiments, at least about 80% of the cellular RNA is maintained as compared to a live control.

[0087] In some embodiments, the total cellular RNAs and proteins of the methanol treated cells are measured by means known to one having skill in the art. In some embodiments, the total cellular RNAs and proteins of the methanol treated cells are measured by high-throughput single-cell analysis. A number of alternative sequencing techniques have been developed andmany are available commercially. These include the use of microarrays of genetic material that can be manipulated so as to permit parallel detection of the ordering of nucleotides in a multitude of fragments of genetic material. The arrays typically include many sites formed or disposed on a substrate. Additional materials, typically single nucleotides or strands of nucleotides (oligonucleotides) are introduced and permitted or encouraged to bind to the template of genetic material to be sequenced, thereby selectively marking the template in a sequence dependent manner. Sequence information may then be gathered by imaging the sites. In certain current techniques, for example, each nucleotide type is tagged with a fluorescent tag or dye that permits analysis of the nucleotide attached at a particular site to be determined by analysis of image data.

[0088] One benefit of the disclosed methods includes the ability for the methanol treated cells to also be used in analysis outside of RNA and protein detection, including sequencing. In some embodiments, the total RNAs in the methanol treated cells are sequenced. In some embodiments, the total protein from the methanol treated cells is sequenced from the DNA sequence conjugated on the at least one antibody. In some embodiments, the methanol treated cells present in single-cell form are prepared using a high throughput method. In some embodiments, the high throughput method utilizes microfluidics. In some embodiments, the method further comprises after step (c), subjecting the cells to single-cell sequencing analysis. In some embodiments, the disclosed methods further comprise, subjecting the cells to single cell sequencing analysis after step (c).

[0089] Many methods, devices and systems are available for sequencing polynucleotides, and can be used for obtaining sequence information of the polynucleotides from the isolated cells in the methods disclosed herein. Droplet-based single-cell RNA sequencing techniques have enabled processing of tens of thousands of cells in a quick and unbiased way with trivial effect on cells. Some non-limiting examples of commercially available systems for high throughput single-cell analysis are Chromium (lOx Genomics), ddSEQ (Illumina and Bio-Rad), scRNA-Seq System running Drop-seq (Dolomite Bio), ICELL8 ex (Takara Bio), MERSCOPE (Vizgen), Rhapsody (BD Biosciences), Evercode (Parse Biosciences), PIPseq (Fluent Biosciences), and Cell DIVE (Leica Microsystems).

[0090] High throughput single-cell sequencing systems generally amplify full-length cDNA using a modified Smart-seq protocol, which incorporates a 5' PCR handle by employing areverse transcriptase’s ability to switch templates at the end of a transcript. Full-length cDNA can be amplified with primers in the 5' tempi ate- switch and 3' poly-T oligonucleotides. Barcoded cDNA ends are further amplified after direct ligation or tagmentation to incorporate sequencing adapters. Amplification bias introduced in the multiple rounds of PCR in these protocols, is mitigated by the incorporation of UMIs. While this is the general methodology of high throughput single-cell sequencing, there are other methods for performing high throughput single-cell sequencing. The methods disclosed herein prepare single cells so that total cellular RNA and total cellular proteins can be analyzed through high throughput single-cell sequencing.Data Analysis

[0091] In some embodiments, the methods include enriching a sample comprising a plurality of cells for cells of interest to produce an enriched cell sample, wherein enriching the sample comprises focusing cells of interest in the sample; isolating one or more cells of interest in the enriched cell sample; and obtaining sequence information of one or more polynucleotides from each of the one or more isolated cells by sequencing the molecularly indexed polynucleotide library. Sequencing the molecularly indexed polynucleotide library can, in some embodiments, include deconvoluting the sequencing result from sequencing the library, possibly using a software-as-a-service platform.Wash Buffer

[0092] In some embodiments, the methanol treated cells are washed after an incubation and before the next step in the process. In some embodiments, wash buffer is used to wash the methanol treated cells. In some embodiments, the wash buffer comprises 0.5-10X PBS and / or 0.01-10% BSA. In some embodiments, the wash buffer comprises 0.5-10X PBS, 0.01-10% BSA, and RNase inhibitor.Blocking Buffer

[0093] The blocking buffer of some embodiments comprises saline sodium citrate (SSC) buffer, DNA blocking agent, reducing agent, and / or RNase inhibitor. The blocking buffer is used to prevent RNA degradation and to improve antibody staining. The components of the blocking buffer allow it to “block” RNA degradation and to “block” antibody conjugate background staining.

[0094] In some embodiments, the blocking buffer comprises SSC buffer. SSC buffer is known in the art and commercially available. SSC buffer can comprise sodium chloride, sodium citrate, and / or trisodium citrate. SSC buffer inhibits RNAse activity which prevents RNA degradation. In some embodiments, a IX formulation of SSC comprises a range of about 25 mM to 300 mM NaCl and a range of about ImM to 50 mM sodium citrate in a pH range of about 6.5 -7.5. In some embodiments, a IX formulation of SSC comprises about 150 mM NaCl and about 15 mM sodium citrate at a pH of about 7.0.

[0095] In some embodiments, the blocking buffer comprises a range of about 0.1X to 30X SSC buffer. In some embodiments, the blocking buffer comprises a range of about 0.5X to 15X SSC buffer. In some embodiments, the blocking buffer comprises a range of about IX to 10X SSC buffer. In some embodiments, the blocking buffer comprises a range of about 2X to 8XSSC buffer. In some embodiments, the blocking buffer comprises a range of about 3X to 6XSSC buffer. In some embodiments, the blocking buffer comprises about IX SSC. In some embodiments, the blocking buffer comprises about 2X SSC. In some embodiments, the blocking buffer comprises about 3X SSC. In some embodiments, the blocking buffer comprises about 4X SSC. In some embodiments, the blocking buffer comprises about 5X SSC. In some embodiments the blocking buffer comprises about 6X SSC. In some embodiments, the blocking buffer comprises about 7X SSC. In some embodiments, the blocking buffer comprises about 8X SSC. In some embodiments, the blocking buffer comprises about 9X SSC. In some embodiments, the blocking buffer comprises about 10X SSC.

[0096] In some embodiments, the blocking buffer comprises a DNA blocking agent. A DNA blocking agent is any agent that minimizes non-specific binding and unwanted background signals, leading to improved antibody staining. DNA blocking agents work by reducing antibody conjugate background. DNA blocking agents are known in the art and are commercially available. In some embodiments, a DNA blocking agent is free DNA from at least one source of DNA, free RNA from at least one source of RNA, single-stranded DNA, doublestranded DNA, transfer RNA (tRNA), ribosomal RNA (rRNA), messenger RNA (mRNA), a combination of free DNA from at least two sources of DNA, a combination of free RNA from at least two sources of RNA, or a combination of free DNA from at least one source of DNA and free RNA from at least one source of RNA. In some embodiments, the free DNA is prepared from highly pure, phenol- chloroform extracted DNA, and DNase-free, RNase-free (DEPC-treated), distilled, deionized water. In some embodiments, the DNA or RNA used for the DNA blocking agent is sourced from several sources. In some embodiments the DNA or RNA used for the DNA blocking agent is from bacteria. Non-limiting examples of free DNA include DNA from calf thymus, salmon sperm, and herring sperm. In some embodiments, the free DNA is sheared.

[0097] In some embodiments, the blocking buffer comprises a range of about 0.1 to 5% DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 0.2 to 4% DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 0.3 to 3% DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 0.4 to 2.5% DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 0.5 to 2% DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 0.6 to 1.9% DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 0.7 to 1.8% DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 0.8 to 1.7% DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 0.9 to 1.6% DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 1.0 to 1.5% DNA blocking agent. In some embodiments, the blocking buffer comprises about 1.0% DNA blocking agent. In some embodiments, the blocking buffer comprises about 1.1% DNA blocking agent. In some embodiments, the blocking buffer comprises about 1.2% DNA blocking agent. In some embodiments, the blocking buffer comprises about 1.3% DNA blocking agent. In some embodiments, the blocking buffer comprises about 1.4% DNA blocking agent. In some embodiments, the blocking buffer comprises about 1.5% DNA blocking agent. In some embodiments, the blocking buffer comprises about 1.6% DNA blocking agent. In some embodiments, the blocking buffer comprises about 1.7% DNA blocking agent. In some embodiments, the blocking buffer comprises about 1.8% DNA blocking agent. In some embodiments, the blocking buffer comprises about 1.9% DNA blocking agent. In some embodiments, the blocking buffer comprises about 2.0% DNA blocking agent. In some embodiments, the blocking buffer comprises about 2.5% DNA blocking agent. In some embodiments, the blocking buffer comprises about 3% DNA blocking agent. In some embodiments, the blocking buffer comprises about 4% DNA blocking agent. In some embodiments, the blocking buffer comprises about 5% DNA blocking agent.

[0098] In some embodiments, the blocking buffer comprises a range of about 1 pg / mL to 5 mg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 10 pg / mL to 4.5 mg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 20 pg / mL to 4 mg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 30 pg / mL to 3.5 mg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 40 pg / mL to 3 mg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 50 pg / mL to 2.5 mg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 75 pg / mL to 2 mg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 100 pg / mL to 1.5 mg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 150 pg / mL to 1 mg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 200 pg / mL to 950 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 250 pg / mL to 900 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 500 pg / mL to 850 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 600 pg / mL to 800 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 700 pg / mL to 750 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 800 pg / mL to 700 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 900 pg / mL to 650 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 1000 pg / mL to 600 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 1000 pg / mL to 550 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 5 pg / mL to 500 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 10 pg / mL to 450 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 20 pg / mL to 400 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 25 pg / mL to 375 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 30 pg / mL to 350 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 35 pg / mL to 325 pg / mLof a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 40 pg / mL to 300 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 45 pg / mL to 275 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 46 pg / mL to 250 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 48 pg / mL to 225 pg / mL of a DNA blocking agent. In some embodiments, the blocking buffer comprises a range of about 50 pg / mL to 200 pg / mL of a DNA blocking agent.

[0099] In some embodiments, the blocking buffer comprises Bovine Serum Albumin (BSA).BSA is known in the art and is commercially available. BSA is often used as a blocking agent to minimize antibody background and minimize non-specific immunostaining. In some embodiments, the blocking buffer comprises a range of about 0.01% to 20% BSA. In some embodiments, the blocking buffer comprises a range of about 0.01% to 18% BSA. In some embodiments, the blocking buffer comprises a range of about 0.01% to 16% BSA. In some embodiments, the blocking buffer comprises a range of about 0.01% to 15% BSA. In some embodiments, the blocking buffer comprises a range of about 0.01% to 14% BSA. In some embodiments, the blocking buffer comprises a range of about 0.01% to 13% BSA. In some embodiments, the blocking buffer comprises a range of about 0.01% to 12% BSA. In some embodiments, the blocking buffer comprises a range of about 0.01% to 11% BSA. In some embodiments, the blocking buffer comprises a range of about 0.01% to 10% BSA. In some embodiments, the blocking buffer comprises a range of about 0.01% to 9% BSA. In some embodiments, the blocking buffer comprises a range of about 0.01% to 8% BSA. In some embodiments, the blocking buffer comprises a range of about 0.01% to 7% BSA. In some embodiments, the blocking buffer comprises a range of about 0.01% to 6% BSA. In some embodiments, the blocking buffer comprises a range of about 0.01% to 5% BSA. In some embodiments, the blocking buffer comprises a range of about 0.02% to 4% BSA. In some embodiments, the blocking buffer comprises a range of about 0.03% to 3% BSA. In some embodiments, the blocking buffer comprises a range of about 0.04% to 2% BSA. In some embodiments, the blocking buffer comprises a range of about 0.05% to 1% BSA. In some embodiments, the blocking buffer comprises a range of about 0.06% to 0.9% BSA. In some embodiments, the blocking buffer comprises a range of about 0.07% to 0.8% BSA. In some embodiments, the blocking buffer comprises a range of about 0.08% to 0.7% BSA. In someembodiments, the blocking buffer comprises a range of about 0.09% to 0.6% BSA. In some embodiments, the blocking buffer comprises a range of about 0.1% to 0.8% BSA. In some embodiments, the blocking buffer comprises about 0.01% BSA. In some embodiments, the blocking buffer comprises about 0.02% BSA. In some embodiments, the blocking buffer comprises about 0.03% BSA. In some embodiments, the blocking buffer comprises about 0.04% BSA. In some embodiments, the blocking buffer comprises about 0.05% BSA. In some embodiments, the blocking buffer comprises about 0.06% BSA. In some embodiments, the blocking buffer comprises about 0.07% BSA. In some embodiments, the blocking buffer comprises about 0.08% BSA. In some embodiments, the blocking buffer comprises about 0.09% BSA. In some embodiments, the blocking buffer comprises about 0.1% BSA. In some embodiments, the blocking buffer comprises about 0.2% BSA. In some embodiments, the blocking buffer comprises about 0.3% BSA. In some embodiments, the blocking buffer comprises about 0.4% BSA. In some embodiments, the blocking buffer comprises about 0.5% BSA. In some embodiments, the blocking buffer comprises about 0.6% BSA. In some embodiments, the blocking buffer comprises about 0.7% BSA. In some embodiments, the blocking buffer comprises about 0.8% BSA. In some embodiments, the blocking buffer comprises about 0.9% BSA. In some embodiments, the blocking buffer comprises about 1% BSA. In some embodiments, the blocking buffer comprises about 2% BSA. In some embodiments, the blocking buffer comprises about 3% BSA. In some embodiments, the blocking buffer comprises about 4% BSA. In some embodiments, the blocking buffer comprises about 5% BSA. In some embodiments, the blocking buffer comprises about 6% BSA. In some embodiments, the blocking buffer comprises about 7% BSA. In some embodiments, the blocking buffer comprises about 8% BSA. In some embodiments, the blocking buffer comprises about 9% BSA. In some embodiments, the blocking buffer comprises about 10% BSA. In some embodiments, the blocking buffer comprises about 12% BSA. In some embodiments, the blocking buffer comprises about 15% BSA. In some embodiments, the blocking buffer comprises about 20% BSA.

[0100] In some embodiments, the blocking buffer comprises a reducing agent. Reducing agents are elements or compounds that donate an electron to an oxidizer compound, hence a compound may be “reduced” (lose an electron) to create an “oxidized” state and the reaction can typically be reversed by “oxidizing” (donating an electron) a compound back into its “reduced”state. Reducing agents inhibit RNAse activity and therefore prevent RNA degradation. At the protein level, reducing agents are often critical in cleaving the disulfide bonds between cysteine amino acids. Reducing agents are known in the art and are commercially available. Some nonlimiting examples of reducing agents include dithiothreitol (DTT), P-mercaptoethanol, di thioerythritol (DTE), L-glutathione (GSH), Tris (2-Carboxyethyl) phosphine hydrochloride (TCEP), cysteine, cysteamine, or salts of sulfurous acid.

[0101] In some embodiments, the blocking buffer comprises about 0.1 to 20% reducing agent. In some embodiments, the blocking buffer comprises about 0.2 to 18% reducing agent. In some embodiments, the blocking buffer comprises about 0.3 to 16% reducing agent. In some embodiments, the blocking buffer comprises about 0.4 to 14% reducing agent. In some embodiments, the blocking buffer comprises about 0.5 to 12% reducing agent. In some embodiments, the blocking buffer comprises about 0.6 to 10% reducing agent. In some embodiments, the blocking buffer comprises about 0.7 to 9% reducing agent. In some embodiments, the blocking buffer comprises about 0.8 to 8% reducing agent. In some embodiments, the blocking buffer comprises about 0.9 to 7% reducing agent. In some embodiments, the blocking buffer comprises about 1 to 6% reducing agent. In some embodiments, the blocking buffer comprises about 2 to 5% reducing agent. In some embodiments, the blocking buffer comprises about 3 to 4% reducing agent. In some embodiments, the blocking buffer comprises about 0.5% reducing agent. In some embodiments, the blocking buffer comprises about 0.6% reducing agent. In some embodiments, the blocking buffer comprises about 0.7% reducing agent. In some embodiments, the blocking buffer comprises about 0.8% reducing agent. In some embodiments, the blocking buffer comprises about 0.9% reducing agent. In some embodiments, the blocking buffer comprises about 1.0% reducing agent. In some embodiments, the blocking buffer comprises about 1.1% reducing agent. In some embodiments, the blocking buffer comprises about 1.2% reducing agent. In some embodiments, the blocking buffer comprises about 1.3% reducing agent. In some embodiments, the blocking buffer comprises about 1.4% reducing agent. In some embodiments, the blocking buffer comprises about 1.5% reducing agent. In some embodiments, the blocking buffer comprises about 1 .6% reducing agent. In some embodiments, the blocking buffer comprises about 1.7% reducing agent. In some embodiments, the blocking buffer comprises about 1.8% reducing agent. In some embodiments, the blocking buffer comprises about 1.9%reducing agent. In some embodiments, the blocking buffer comprises about 2% reducing agent. In some embodiments, the blocking buffer comprises about 3% reducing agent. In some embodiments, the blocking buffer comprises about 4% reducing agent. In some embodiments, the blocking buffer comprises about 5% reducing agent. In some embodiments, the blocking buffer comprises about 6% reducing agent. In some embodiments, the blocking buffer comprises about 7% reducing agent. In some embodiments, the blocking buffer comprises about 8% reducing agent. In some embodiments, the blocking buffer comprises about 9% reducing agent. In some embodiments, the blocking buffer comprises about 10% reducing agent. In some embodiments, the blocking buffer comprises about 12% reducing agent. In some embodiments, the blocking buffer comprises about 14% reducing agent. In some embodiments, the blocking buffer comprises about 16% reducing agent. In some embodiments, the blocking buffer comprises about 18% reducing agent. In some embodiments, the blocking buffer comprises about 20% reducing agent.

[0102] In some embodiments, the blocking buffer comprises an RNase inhibitor. RNase inhibitors are compounds intended to inactivate ribonuclease enzymes, which degrade RNA. RNase inhibitors are known in the art and many are commercially available. Non-limiting examples of RNase inhibitors include Guanidinium thiocyanate (GTC) or guanidinium isothiocyanate (GITC).

[0103] In some embodiments, the blocking buffer comprises a range of about 0.1 to 10% RNase inhibitor. In some embodiments, the blocking buffer comprises a range of about 0.2 to 9% RNase inhibitor. In some embodiments, the blocking buffer comprises a range of about 0.3 to 8% RNase inhibitor. In some embodiments, the blocking buffer comprises a range of about 0.4 to 7% RNase inhibitor. In some embodiments, the blocking buffer comprises a range of about 0.5 to 6% RNase inhibitor. In some embodiments, the blocking buffer comprises a range of about 0.6 to 5% RNase inhibitor. In some embodiments, the blocking buffer comprises a range of about 0.7 to 4% RNase inhibitor. In some embodiments, the blocking buffer comprises a range of about 0.8 to 3% RNase inhibitor. In some embodiments, the blocking buffer comprises a range of about 0.9 to 2% RNase inhibitor. In some embodiments, the blocking buffer comprises 0.1% RNase inhibitor. In some embodiments, the blocking buffer comprises about 0.2% RNase inhibitor. In some embodiments, the blocking buffer comprises 0.3% RNase inhibitor. In some embodiments, the blocking buffer comprises about 0.4% RNase inhibitor. Insome embodiments, the blocking buffer comprises 0.5% RNase inhibitor. In some embodiments, the blocking buffer comprises about 0.6% RNase inhibitor. In some embodiments, the blocking buffer comprises 0.7% RNase inhibitor. In some embodiments, the blocking buffer comprises about 0.8% RNase inhibitor. In some embodiments, the blocking buffer comprises 0.9% RNase inhibitor. In some embodiments, the blocking buffer comprises about 1.0 % RNase inhibitor. In some embodiments, the blocking buffer comprises 1.1% RNase inhibitor. In some embodiments, the blocking buffer comprises about 1.2% RNase inhibitor. In some embodiments, the blocking buffer comprises 1.3% RNase inhibitor. In some embodiments, the blocking buffer comprises about 1.4% RNase inhibitor. In some embodiments, the blocking buffer comprises 1.5% RNase inhibitor. In some embodiments, the blocking buffer comprises about 2.0% RNase inhibitor. In some embodiments, the blocking buffer comprises about 3.0% RNase inhibitor. In some embodiments, the blocking buffer comprises about 4.0% RNase inhibitor, In some embodiments, the blocking buffer comprises about 5.0% RNase inhibitor, In some embodiments, the blocking buffer comprises about 6.0% RNase inhibitor, In some embodiments, the blocking buffer comprises about 7.0% RNase inhibitor, In some embodiments, the blocking buffer comprises about 8.0% RNase inhibitor, In some embodiments, the blocking buffer comprises about 9.0% RNase inhibitor, In some embodiments, the blocking buffer comprises about 10% RNase inhibitor.Antibody Reagent

[0104] Step (c) of some embodiments requires incubation of the methanol treated cells in an antibody reagent. In some embodiments, the antibody reagent comprises phosphate buffered saline (PBS), DNA blocking agent, bovine serum albumin (BSA), nuclease free water, at least one antibody, and RNase inhibitor.

[0105] In some embodiments, the antibody reagent comprises PBS. In some embodiments, the antibody reagent comprises a range of about 0. IX to 20X PBS. In some embodiments, the antibody reagent comprises a range of about 0.2X to 18X PBS. In some embodiments, the antibody reagent comprises a range of about 0.3X to 16X PBS. In some embodiments, the antibody reagent comprises a range of about 0.4X to 14X PBS. In some embodiments, the antibody reagent comprises a range of about 0.5X to 12X PBS. In some embodiments, the antibody reagent comprises a range of about 0.6X to 10X PBS. In some embodiments, theantibody reagent comprises a range of about 0.7X to 9X PBS. Tn some embodiments, the antibody reagent comprises a range of about 0.8X to 8X PBS. In some embodiments, the antibody reagent comprises a range of about 0.9X to 7X PBS. In some embodiments, the antibody reagent comprises a range of about IX to 6X PBS. In some embodiments, the antibody reagent comprises a range of about 2X to 5X PBS. In some embodiments, the antibody reagent comprises about 0.5X PBS. In some embodiments, the antibody reagent comprises about IX PBS. In some embodiments, the antibody reagent comprises about 1.5X PBS. In some embodiments, the antibody reagent comprises about 2X PBS. In some embodiments, the antibody reagent comprises about 2.5X PBS. In some embodiments, the antibody reagent comprises about 3X PBS. In some embodiments, the antibody reagent comprises about 3.5X PBS. In some embodiments, the antibody reagent comprises about 4X PBS. In some embodiments, the antibody reagent comprises about 4.5X PBS. In some embodiments, the antibody reagent comprises about 5X PBS. In some embodiments, the antibody reagent comprises about 5.5X PBS. In some embodiments, the antibody reagent comprises about 6X PBS. In some embodiments, the antibody reagent comprises about 6.5X PBS. In some embodiments, the antibody reagent comprises about 7X PBS. In some embodiments, the antibody reagent comprises about 7.5X PBS. In some embodiments, the antibody reagent comprises about 8X PBS. In some embodiments, the antibody reagent comprises about 9X PBS. In some embodiments, the antibody reagent comprises about 10X PBS.

[0106] In some embodiments, the antibody reagent comprises DNA blocking agent. This can be the same or different DNA blocking agent as used in the blocking buffer described above. In some embodiments, the antibody reagent comprises a range of about 0.1 pg / mL to 1000 pg / mL DNA blocking agent. In some embodiments, the antibody reagent comprises a range of about 1 pg / mL to 900 pg / mL DNA blocking agent. In some embodiments, the antibody reagent comprises a range of about 10 pg / mL to 800 pg / mL DNA blocking agent. In some embodiments, the antibody reagent comprises a range of about 50 pg / mL to 700 pg / mL DNA blocking agent. In some embodiments, the antibody reagent comprises a range of about 60 pg / mL to 600 pg / mL DNA blocking agent. In some embodiments, the antibody reagent comprises a range of about 70 pg / mL to 500 pg / mL DNA blocking agent. In some embodiments, the antibody reagent comprises a range of about 80 pg / mL to 400 pg / mL DNA blocking agent. In some embodiments, the antibody reagent comprises a range of about 90 pg / mL to 300 pg / mL DNAblocking agent. In some embodiments, the antibody reagent comprises a range of about 50 pg / mL to 200 pg / mL DNA blocking agent. In some embodiments, the antibody reagent comprises a range of about 100 pg / mL to 250 pg / mL DNA blocking agent. In some embodiments, the antibody reagent comprises a range of about 100 pg / mL to 200 pg / mL DNA blocking agent. In some embodiments, the antibody reagent comprises a range of about 1000 pg / mL to 150 pg / mL DNA blocking agent.

[0107] In some embodiments, the antibody reagent comprises BSA. In some embodiments, the antibody reagent comprises a range of about 0.01 to 10% BSA. In some embodiments, the antibody reagent comprises a range of about 0.05 to 9% BSA. In some embodiments, the antibody reagent comprises a range of about 0.1 to 8% BSA. In some embodiments, the antibody reagent comprises a range of about 0.2 to 7% BSA. In some embodiments, the antibody reagent comprises a range of about 0.3 to 6% BSA. In some embodiments, the antibody reagent comprises a range of about 0.4 to 5% BSA. In some embodiments, the antibody reagent comprises a range of about 0.5 to 4% BSA. In some embodiments, the antibody reagent comprises a range of about 0.6 to 3% BSA. In some embodiments, the antibody reagent comprises a range of about 0.7 to 2% BSA. In some embodiments, the antibody reagent comprises a range of about 0.8 to 1.5% BSA. In some embodiments, the antibody reagent comprises a range of about 0.9 to 1.5% BSA. In some embodiments, the antibody reagent comprises a range of about 1 to 1.5% BSA. In some embodiments, the antibody reagent comprises a range of about 0.8 to 1.4% BSA. In some embodiments, the antibody reagent comprises a range of about 0.8 to 1.3% BSA. In some embodiments, the antibody reagent comprises a range of about 0.8 to 1.2% BSA.

[0108] In some embodiments, the antibody reagent comprises at least one antibody. In some embodiments, the antibody reagent comprises 2 or more antibodies. In some embodiments, the antibody reagent comprises 3 or more antibodies. In some embodiments, the antibody reagent comprises 4 or more antibodies. In some embodiments, the antibody reagent comprises 5 or more antibodies. In some embodiments, the antibody reagent comprises 6 or more antibodies. In some embodiments, the antibody reagent comprises 7 or more antibodies. In some embodiments, the antibody reagent comprises 8 or more antibodies. In some embodiments, the antibody reagent comprises 9 or more antibodies. In some embodiments, the antibody reagent comprises 10 or more antibodies. In some embodiments, the antibody reagent comprises 12 ormore antibodies. In some embodiments, the antibody reagent comprises 15 or more antibodies. In some embodiments, the antibody reagent comprises 18 or more antibodies. In some embodiments, the antibody reagent comprises 20 or more antibodies. In some embodiments, the antibody reagent comprises 25 or more antibodies. In some embodiments, the antibody reagent comprises 30 or more antibodies. In some embodiments, the antibody reagent comprises 35 or more antibodies. In some embodiments, the antibody reagent comprises 40 or more antibodies. In some embodiments, the antibody reagent comprises 45 or more antibodies. In some embodiments, the antibody reagent comprises 50 or more antibodies. In some embodiments, the antibody reagent comprises 55 or more antibodies. In some embodiments, the antibody reagent comprises 60 or more antibodies. In some embodiments, the antibody reagent comprises 65 or more antibodies. In some embodiments, the antibody reagent comprises 70 or more antibodies. In some embodiments, the antibody reagent comprises 75 or more antibodies. In some embodiments, the antibody reagent comprises 80 or more antibodies. In some embodiments, the antibody reagent comprises 90 or more antibodies. In some embodiments, the antibody reagent comprises 100 or more antibodies. In some embodiments, the antibody reagent comprises 150 or more antibodies.

[0109] In some embodiments, the antibody reagent comprises a range of about 0.001 pg / mL to 500 pg / mL of at least one antibody. In some embodiments, the antibody reagent comprises a range of about 0.001 pg / mL to 400 pg / mL of at least one antibody. In some embodiments, the antibody reagent comprises a range of about 0.001 pg / mL to 300 pg / mL of at least one antibody. In some embodiments, the antibody reagent comprises a range of about 0.001 pg / mL to 200 pg / mL of at least one antibody. In some embodiments, the antibody reagent comprises a range of about 0.001 pg / mL to 100 pg / mL of at least one antibody. In some embodiments, the antibody reagent comprises a range of about 0.001 pg / mL to 75 pg / mL of at least one antibody. In some embodiments, the antibody reagent comprises a range of about 0.005 pg / mL to 50 pg / mL of at least one antibody. In some embodiments, the antibody reagent comprises a range of about 0.01 pg / mL to 25 pg / mL of at least one antibody. In some embodiments, the antibody reagent comprises a range of about 0.015 pg / mL to 15 pg / mL of at least one antibody. In some embodiments, the antibody reagent comprises a range of about 0.02 pg / mL to 10 pg / mL of at least one antibody. In some embodiments, the antibody reagent comprises a range of about 0.021 pg / mL to 9 pg / mL of at least one antibody. In some embodiments, the antibody reagentcomprises a range of about 0.022 pg / mL to 8 ng / mE of at least one antibody. In some embodiments, the antibody reagent comprises a range of about 0.023 pg / mL to 7 pg / mL of at least one antibody. In some embodiments, the antibody reagent comprises a range of about 0.024 pg / mL to 6 pg / mL of at least one antibody. In some embodiments, the antibody reagent comprises a range of about 0.025 pg / mL to 5 pg / mL of at least one antibody.

[0110] In some embodiments, the at least one antibody of the antibody reagent is an antibody to a protein of interest. In some embodiments, the antibody reagent is an antibody to a post- translational modification of a protein of interest. In some embodiments, the cellular proteins include an individual target protein, a protein complex, a post-translational modification in a protein, and / or a protein / nucleic acid complex. In some embodiments, the detection of proteins can include the detection of the level of proteins and / or post translational modifications of proteins. In some embodiments, the antibody conjugate can detect certain post-translational modifications of target proteins. In some embodiments, the detecting includes levels of proteins (quantity), identity of and post-translational modification of a protein, including but not limited to, methylation, acetylation, glycosylation, phosphorylation, and ubiquitination. Protein targets include peptides, enzymes, hormones, structural components such as viral capsid proteins, and antibodies. Protein targets may be synthetic or derived from naturally occurring sources. An individual protein is an isolated polypeptide chain. A protein complex includes two or more polypeptide chains. Samples may include proteins with post translational modifications including but not limited to phosphorylation, methionine oxidation, deamidation, glycosylation, ubiquitination, carb amyl ati on, S-carboxymethylation, acetylation, and methylation. Methods of the disclosure work with any target protein. Non-limiting examples of targets include: T- bet / TBX21, CD3 (UCHT1), CD8a (SKl), S100A9, CD4 (RPA-T4), TCF1 / TCF7, Ikaros, Aiolos, CD19, Ibal / AIF-1, Phospho-CREB, CREB, Phospho-Akt, Akt, phospho-p44 / 42 MAPK (Erkl / 2), p44 / 42 MAPK (Erkl / 2), Vimentin, phospho-stat3, stat3, phospho-stat4, stat4, phospho- statl, statl, NCAM1, Phospho-Histone H3, histone H3, acetyl -hi stone H3, tri-methyl-histone H3, GAPDH, Mouse (G3A1) mAb IgGl Isotype Control, Phospho-S6 Ribosomal Protein, S6 ribosomal protein, phospho-4E-BPl, CD68, CDl lb / ITGAM, IFI16, phosphor-Zap-70, Zap-70, NF ATI, BATF, phospho-glucocorticoid receptor, glucocorticoid receptor, Phospho-PLCyl , PLCyl, Phospho-IRF-3, IRF-3, NF-KB p65, phospho-NF-KB p65, FoxOl, PLCyl, phospho- cJun, eJun, IL-17F, IL-17A, caspase-3, phospho-histone H2A.X, histone H2A.X, phospho-YAP,YAP, phospho-p38 MAPK, p38 MAPK, phospho-Rb, Rb (4H1), NRF2, phospho-PTEN, PTEN, PCNA, SMAD2 / 3, phospho- EGF receptor, EGF receptor, c-Myc / N-Myc, phospho- SMAD2, ATF-4, phospho-mTOR, mTOR, phospho-eIF2a, phospho-TBKl / NAK, eIF2a, TBK1 / NAK, Phospho-SAPK / INK, SAPK / INK, C0L1A1, phospho-c-Fos, c-Fos, Phospho-AMPKa, AMPKa, phospho- Stat6, Stat6, and E-cadherin. In some embodiments, the at least one antibody is an antibody directed to T-bet / TBX21, CD3 (UCHT1), CD8a (SKI), S100A9, CD4 (RPA-T4), TCF1 / TCF7, Ikaros, Aiolos, CD19, Ibal / AIF-1, Phospho-CREB, CREB, Phospho- Akt, Akt, phospho-p44 / 42 MAPK (Erkl / 2), p44 / 42 MAPK (Erkl / 2), Vimentin, phospho-stat3, stat3, phospho-stat4, stat4, phospho-statl, statl, NCAM1, Phospho-Histone H3, histone H3, acetylhistone H3, tri-methyl-histone H3, GAPDH, Mouse (G3A1) mAb IgGl Isotype Control, Phospho-S6 Ribosomal Protein, S6 ribosomal protein, phospho-4E-BPl, CD68, CDl lb / ITGAM, IFI16, phosphor-Zap-70, Zap-70, NF ATI, BATF, phospho-glucocorticoid receptor, glucocorticoid receptor, Phospho-PLCyl, PLCyl, Phospho-IRF-3, IRF-3, NF-KB p65, phospho- NF-KB p65, FoxOl, PLCyl, phospho-cJun, eJun, IL-17F, IL-17A, caspase-3, phospho-histone H2A.X, histone H2A.X, phospho-YAP, YAP, phospho-p38 MAPK, p38 MAPK, phospho-Rb, Rb (4H1), NRF2, phospho-PTEN, PTEN, PCNA, SMAD2 / 3, phospho- EGF receptor, EGF receptor, c-Myc / N-Myc, phospho- SMAD2, ATF-4, phospho-mTOR, mTOR, phospho-eIF2a, phospho-TBKl / NAK, eIF2a, TBK1 / NAK, Phospho-SAPK / INK, SAPK / INK, C0L1A1, phospho-c-Fos, c-Fos, Phospho-AMPKa, AMPKa, phospho-Stat6, Stat6, and E-cadherin.[OUl] In some embodiments, the at least one antibody of the antibody reagent is a conjugated antibody. A conjugated antibody (also known as a tagged, loaded or labeled antibody) is a polyclonal or monoclonal antibody that has a molecule attached which can be used to create a detectable signal. Detection can be visualized by color-generation, fluorescence, or other signals. Conjugated antibodies are known in the art and are commercially available. Methods of making conjugated antibodies are known in the art. Kits for conjugating antibodies are available in the art.

[0112] In some embodiments, the conjugated antibody is conjugated to a nucleic acid sequence. In some embodiments, the nucleic acid ranges from about 5 to about 200 nucleotides in length. In some embodiments, the nucleic acid ranges from about 10 to about 150 nucleotides in length. In some embodiments, the nucleic acid ranges from about 15 to about 125 nucleotides in length. In some embodiments, the nucleic acid ranges from about 20 to about 100 nucleotidesin length. In some embodiments, the nucleic acid ranges from about 25 to about 100 nucleotides in length. In some embodiments, the nucleic acid ranges from about 20 to about 75 nucleotides in length. In some embodiments, the nucleic acid ranges from about 20 to about 50 nucleotides in length. In some embodiments, the nucleic acid is at least 5 nucleotides in length. In some embodiments, the nucleic acid is at least 10 nucleotides in length. In some embodiments, the nucleic acid is at least 11 nucleotides in length. In some embodiments, the nucleic acid is at least 12 nucleotides in length. In some embodiments, the nucleic acid is at least 13 nucleotides in length. In some embodiments, the nucleic acid is at least 13 nucleotides in length. In some embodiments, the nucleic acid is at least 14 nucleotides in length. In some embodiments, the nucleic acid is at least 15 nucleotides in length. In some embodiments, the nucleic acid is at least 16 nucleotides in length. In some embodiments, the nucleic acid is at least 17 nucleotides in length. In some embodiments, the nucleic acid is at least 18 nucleotides in length. In some embodiments, the nucleic acid is at least 19 nucleotides in length. In some embodiments, the nucleic acid is at least 20 nucleotides in length.

[0113] In some embodiments, the nucleotides comprise a capture sequence and barcode sequence. In some embodiments, the nucleotides comprise a capture sequence, barcode sequence, and Unique Molecular Identifier (UMI) sequence. In some embodiments, the capture sequence comprises sequences that are specific to each commercially available protocol. Nonlimiting examples of such protocols are Chromium (lOx Genomics), ddSEQ (Illumina and BioRad), scRNA-Seq System running Drop-seq (Dolomite Bio), ICELL8 ex (Takara Bio), MERSCOPE (Vizgen), Rhapsody (BD Biosciences), Evercode (Parse Biosciences), PIPseq (Fluent Biosciences), and Cell DIVE (Leica Microsystems). In some embodiments, the nucleotides comprise an anchor sequence. In some embodiments, the anchor sequence is a polyadenylation sequence. In some embodiments, the anchor sequence is a polyT sequence. In some embodiments, the anchor sequence is a polyG sequence.

[0114] In some embodiments the conjugated antibody is conjugated to nucleic acid sequence that is a DNA sequence. In some embodiments, the conjugated antibody is conjugated to nucleic acid sequence that is a single stranded DNA (ssDNA). In some embodiments, the conjugated antibody is conjugated to a nucleic acid sequence that is an RNA sequence.

[0115] In some embodiments, the antibody reagent comprises an RNase inhibitor. In some embodiments, the RNase inhibitor is the same as the RNase inhibitor used in the blocking buffer described above. In some embodiments, the RNase inhibitor is different than the one used in the blocking buffer described above.

[0116] In some embodiments, the antibody reagent comprises a range of about 0.1 to 10% RNase inhibitor. In some embodiments, the antibody reagent comprises a range of about 0.2 to 9% RNase inhibitor. In some embodiments, the antibody reagent comprises a range of about 0.3 to 8% RNase inhibitor. In some embodiments, the antibody reagent comprises a range of about 0.4 to 7% RNase inhibitor. In some embodiments, the antibody reagent comprises a range of about 0.5 to 6% RNase inhibitor. In some embodiments, the antibody reagent comprises a range of about 1 to 5% RNase inhibitor. In some embodiments, the antibody reagent comprises a range of about 1 to 4% RNase inhibitor. In some embodiments, the antibody reagent comprises a range of about 2 to 3% RNase inhibitor.Sequencing readout.

[0117] After attachment of the labels to the targets in a stochastic manner, the targets may be amplified according to any of the methods disclosed herein and known in the art. The amplification product may be subjected to any available sequencing method known in the art.

[0118] A number of alternative sequencing techniques have been developed and many are available commercially. These include the use of microarrays of genetic material that can be manipulated so as to permit parallel detection of the ordering of nucleotides in a multitude of fragments of genetic material. The arrays typically include many sites formed or disposed on a substrate. Additional materials, typically single nucleotides or strands of nucleotides (oligonucleotides) are introduced and permitted or encouraged to bind to the template of genetic material to be sequenced, thereby selectively marking the template in a sequence dependent manner. Sequence information may then be gathered by imaging the sites. In certain current techniques, for example, each nucleotide type is tagged with a fluorescent tag or dye that permits analysis of the nucleotide attached at a particular site to be determined by analysis of image data.

[0119] In some embodiments, after the completion of step (c), the method further comprises subjecting the cells to single-cell sequencing analysis. Single-cell sequencing examines the nucleic acid sequence information from individual cells with optimized next-generationsequencing technologies, providing a higher resolution of cellular differences and a better understanding of the function of an individual cell in the context of its microenvironment.High-throughput single-cell analysis

[0120] Many methods, devices and systems are available for sequencing polynucleotides, and can be used for obtaining sequence information of the polynucleotides from the isolated cells in the methods disclosed herein. Droplet-based single-cell RNA sequencing techniques have enabled processing of tens of thousands of cells in a quick and unbiased way with trivial effect on cells. Some non-limiting examples of commercially available systems for high throughput single-cell analysis are Chromium (lOx Genomics), ddSEQ (Illumina and Bio-Rad), scRNA-Seq System running Drop-seq (Dolomite Bio), ICELL8 ex (Takara Bio), MERSCOPE (Vizgen), Rhapsody (BD Biosciences), Evercode (Parse Biosciences), PIPseq (Fluent Biosciences), and Cell DIVE (Leica Microsystems).

[0121] High throughput single-cell sequencing systems generally amplify full-length cDNA using a modified Smart-seq protocol, which incorporates a 5' PCR handle by employing a reverse transcriptase’s ability to switch templates at the end of a transcript. Full-length cDNA can be amplified with primers in the 5' template-switch and 3' poly-T oligonucleotides.Barcoded cDNA ends are further amplified after direct ligation or tagmentation to incorporate Illumina sequencing adapters. ddSEQ contains a single amplification step during adapter incorporation after second strand synthesis without amplification of full-length cDNA.Amplification bias introduced in the multiple rounds of PCR in these protocols, is mitigated by the incorporation of UMIs.Data Analysis

[0122] In some embodiments, the methods include enriching a sample comprising a plurality of cells for cells of interest to produce an enriched cell sample, wherein enriching the sample comprises focusing cells of interest in the sample; isolating one or more cells of interest in the enriched cell sample; and obtaining sequence information of one or more polynucleotides from each of the one or more isolated cells by sequencing the molecularly indexed polynucleotide library. Sequencing the molecularly indexed polynucleotide library can, in some embodiments, include deconvoluting the sequencing result from sequencing the library, possibly using a software-as-a-service platform.EXAMPLES

[0123] Example 1. A 4-Step Protocol

[0124] The disclosed method was used to generate single-cell RNA analysis along with simultaneous wholistic single-cell protein analysis. After obtaining a biological tissue sample, the tissue sample was dissociated to release the cells from the tissue. About 1 million to 5 million cells from the dissociated tissue were placed into a microtubule and centrifuged at 300 x g for 5 minutes at 4°C. The supernatant was removed, and the cells washed with lOmL of ice- cold IX phosphate buffered saline (PBS) in a 15mL tube. The cells were then centrifuged at 300 x g for 5 minutes at 4°C. The supernatant was removed without disturbing the cell pellet and the cells were resuspended in 0.5mL of ice-cold IX PBS. The 15mL tube was placed on a benchtop vortex mixer at low speeds so that the sample was not vortexed but was constantly mixed. While the 15mL tube was on the vortex mixer, 4.5mL of ice cold 100% methanol was added to the sample in a dropwise manner. Once the addition of methanol was complete, the sample was incubated in methanol overnight at -20°C.

[0125] The next day, 13pL of DNA blocking agent was combined with 117pL of nuclease free water and the combination was heated at 95°C for 5 minutes. After 5 minutes, the water / DNA blocking agent combination was spun down briefly and placed on ice. Then, 150pL of 20X saline sodium citrate (SSC) buffer, 4pL of 10% bovine serum albumin (BSA), 706pL of Molecular grade / nuclease free water, 40 pL dithiothreitol (DTT), and 2 pL RNase inhibitor were added to the water / DNA blocking agent combination to make blocking buffer. The blocking buffer was mixed by pipetting about 5 times and then was stored on ice.

[0126] Next the sample incubated in methanol was retrieved and centrifuged 850 x g for 5 minutes at 4°C. The supernatant was removed. 1 mL of the blocking buffer was added to the cells and the cell pellet was resuspended in the blocking buffer. The resuspended cells in the blocking buffer were incubated on ice for 30 minutes.

[0127] The antibody reagent was prepared by combining DNA blocking agent with nuclease free water and heating at 95°C for 5 minutes. After 5 minutes, the water / DNA blocking agent combination was spun down briefly and placed on ice. Then 5 uL 20x PBS, 55 uL of 10% BSA and RNase inhibitor were combined with the water / DNA blocking agent combination. Then atleast one antibody was added to the mixture to create the antibody reagent. The antibody reagent was then pipette mixed about 5 times and stored on ice.

[0128] After the resuspended cells had incubated in the blocking buffer, 3 mL of lx PBS w / 0.5% BSA was added. The cells were then filtered through a sterile 40 micron cell strainer into a 50 mL tube and the flow through / filtered cells were transferred into a new 15 mL tube. The cells were then centrifuged at 850 x g for 5 minutes at 4°C. The supernatant was removed without allowing the cell pellet to dry. IOOUL of the antibody reagent was then added to the cell pellet and gently pipetted to resuspend the cells. The cells were incubated in the antibody reagent overnight.

[0129] After the overnight incubation, a wash buffer comprising lx PBS with 0.5% BSA and RNase inhibitor was added to the cells. The cells were resuspended in the wash buffer. The resuspended cells were centrifuged at 850 x g for 5 minutes at 4°C, supernatant removed, and cells resuspended in wash buffer. The resuspended cells were centrifuged at 850 x g for 5 minutes at 4°C. The supernatant was removed, and the cells were again resuspended in wash buffer. The cells were then filtered through a sterile 40-micron filter. The filtered cells were centrifuged at 850 x g for 5 minutes at 4°C, supernatant removed, and cells resuspended in wash buffer. The cells were counted and then processed using a commercially available 10X Genomics Next GEM Single Cell Kit according to the instructions.

[0130] Example 2. Protein Quantification in single-cell RNA sequencing

[0131] Commercially available lOx Genomics 3’ single cell kits were used to validate the disclosed methodology. The cells were prepared using the 4-step process from example 1 above, briefly, single cells were fixed with methanol overnight; the cells were then blocked in blocking buffer; the methanol treated cells were then incubated in a blocking buffer; the methanol treated cells were then treated with an antibody reagent; and then cellular RNAs and target proteins were analyzed.

[0132] To show that the disclosed methods preserve the heterogeneity of peripheral blood mononuclear cells (PBMCs), live PBMCs were collected and divided into two factions. One faction was subjected to the disclosed method of fixation and treatment and compared to a control of live PBMCs. The live control sample (FIG. 3B) and the cells processed through the disclosed method (FIG. 3C) look the same. The percent cell distribution was also remarkablysimilar in both conditions, showing that the disclosed methods preserve the heterogeneity in PBMCs.

[0133] To assess the quality of the disclosed methods regarding RNA and protein, RNA expression of a target and protein expression of the same target was measured and compared (FIG. 4B-U). While RNA expression is well detected, protein expression is more uniform than RNA expression. This emphasizes one strength of the disclosed technology, offering a more accurate representation of the target expression at the protein level. FIG. 4B-4U show how the disclosed technology can display very robust RNA and protein signature, especially when correlated in a single cell dataset.

[0134] However, RNA does not always correlate with protein levels. In order to see the correlation, levels of RNA and protein were measured in a number of samples looking at RNA, protein, and post-translational modifications of protein in the same targets. In experiments conducted comparing RNA to total protein, the correlation is high and close to 1 (results not shown). However, in similar experiments conducted comparing RNA to a protein target having post-translational modification, the correlation between is close to 0, showing a lack of correlation (results not shown). Such a lack of correlation was somewhat expected as these modifications happen at the protein level and cannot be reflected from RNA expression alone. This is also a prime example of how the disclosed methods can be used to uncover missing information in a single cell RNA experiment and offer new biological insights at the protein level, including the post-translational modification level.

[0135] To demonstrate the ability of the disclosed technology to identify cell states which are difficult to do through analyzing RNA alone, CD8 positive T cells were selected from PBMCs. The data was re-analyzed and three clusters of cells were found and labeled as “naive” CD4 T cells, “effector” CD4 cells, and “memory-like” CD4 cells. RNA levels for selected targets did not show a clear picture of the different cellular states. However, protein expression was easily seen. FIG. 5H shows selected CD8 positive T cells from PBMCs, where the data is reanalyzed into three clusters labeled as “naive” CD8 T cells, “effector” CD8 cells, and “memory-like” CD8 cells. FIG. 51, 5K, and 5M show mapped targeted RNA for three different targets, Stat 3, TCF7, and TBX21 RNA, while FIG. 5 J, 5L, and 5N show mapped targeted protein for three different protein targets, phosphorylated STAT3-Ser727, TCF1 / TCF7, and TBX21. TCF7 protein ishighly expressed in the naive state of T cells, which corroborates with previously published papers. Conversely, TBX21 protein was upregulated in the effector / differentiated state. FIG. 5A-5N show that analyzing the RNA level for these targets does not show a clear picture of the different cellular states but how the intracellular protein and post translational modification readout from an experiment performed through the disclosed methods offers a new perspective when identifying cellular states.

[0136] Next, to test whether post-translational modifications can be accurately measured using signaling pathway antibodies and the disclosed methods, Jurkat cells were inhibited with Wortmannin and LY294002. Wortmannin and LY294002 are known to be highly selective and effective inhibitors of the phosphatidylinositol 3 kinase (PI3K) pathway. Akt protein levels were measured as Akt is known to be downstream of PI3K in the PI3K signaling pathway. Total Akt protein levels were identical in both control and drug inhibited Jurkat cells (FIG. 6B). However, Akt protein phosphorylated at Ser473 levels were drastically lower in the drug inhibited Jurkat cells as compared to control Jurkat cells (FIG. 6C). Additionally, S6 protein levels were measured as S6 is known to be downstream of Akt in the PI3K pathway. The level of S6 protein remained the same in both control and drug inhibited Jurkat cells (FIG. 6D) while phosphorylated S6 protein was inhibited in the cells treated with PI3K inhibitors (FIG. 6E). This data offers a proof of concept showing that the disclosed methods can viably measure post- translational modifications in a single-cell assay.

[0137] Next, primary T cells were stimulated with PMA and lonomycin to promote phosphorylation levels since phosphorylation levels are generally low in primary T cells (FIG. 7A and 7B). After stimulation, the T cells were then split into a control and experimental group where the experimental group were further treated with a phosphatase to dephosphorylate protein residues (FIG. 8A-P). While total protein levels were not affected through the phosphatase treatment (FIG. 8B, 8D, 8F, 8H, 8J, 8L, 8N, and 8P), the experimental groups showed that the phosphorylation of the proteins were significantly decreased where the cells had been stimulated and then treated with phosphatase (FIG. 8A, 8C, 8E, 8G, 81, 8K, 8M, and 80). This data showed that post-translational modifications of proteins can be quantified in both primary cells and cell lines.

[0138] Lastly, PBMCs were obtained and cultured in a control group and experimental group where the experimental PBMCs were stimulated lipopolysaccharide for 3 consecutive days. The data was analyzed through the generation of heatmaps showing how levels of phosphorylated Stat3 Ser 727 were impacted before and after lipopolysaccharide treatment. RNA or protein targets correlating with the changes of phospho-stat3 in each cell type was measured (FIG. 2A). For example, there was an increase in phospho-stat3 (727) in monocytes and that increase was followed by an increase or decrease in certain RNA and protein targets (FIG. 2A). Stat3 is known to translocate to the nucleus following phosphorylation and acts as a transcription factor. As such, it can be determined which targets are directly impacted by phospho-Stat3 through analyzing Chip-seq datasets. This is just one example of how the disclosed methods allow a deeper understanding of multiple conditions from heterogenous and complex samples.

[0139] Once the promoter regions bound by stat3 are determined, the molecular mechanism in each cell type can be deconvoluted for the stimulation, inhibition, knockout, knockdown, or whatever experimental conditions are chosen. This process can start by measuring surface, intracellular, and nuclear proteins, as well as post-translational modifications of those proteins. All of this can be measured while also quantifying RNA in single cells.

[0140] The disclosed methods are also a great screening tool to understand which signaling pathway and RNA targets are impacted in different cell types. Such knowledge will help guide the research and narrow targets as to what the next targets will be.

[0141] The disclosed methods can also be employed as a great hypothesis generation tool when trying to understand the molecular mechanism in different biological conditions.

Claims

WHAT IS CLAIMED IS:

1. A method of single-cell analysis of total cellular ribonucleic acids (RNAs) and one or more proteins, the method comprising:(a) incubating cells present in single-cell form in methanol;(b) incubating the methanol treated cells in a blocking buffer;(c) incubating the methanol treated cells with an antibody reagent; and(d) detecting total cellular RNAs and one or more proteins in the cells.

2. The method of claim 1, wherein the cells are incubated in methanol for at least 10 minutes.

3. The method of claim 2, wherein the cells are incubated in methanol for at least 30 minutes.

4. The method of claim 2, wherein the cells are incubated in methanol at least overnight.

5. The method of any one of the preceding claims, wherein the cells are incubated in methanol at a temperature ranging from -100°C to room temperature.

6. The method of any one of the preceding claims, wherein the cells are incubated in methanol at -20°C.

7. The method of any one of the preceding claims, wherein the cells are incubated in methanol at 4°C.

8. The method of any one of the preceding claims, wherein the cells are incubated in methanol at -80°C.

9. The method of any one of the preceding claims, wherein the methanol is a molecular grade methanol that is at least 50% methanol, at least 70% methanol; at least 80% methanol, at least 90% methanol, at least 95% methanol, or 100% methanol.

10. The method of any one of the preceding claims, further comprising removing methanol after step (a) and resuspending the methanol treated cells in the blocking buffer; then incubating as in step (b).

11. The method of any one of the preceding claims, wherein steps (b) and (c) are performed simultaneously.

12. The method of any one of claims 1-10, wherein the method comprises step (b) incubating the methanol treated cells in a blocking buffer wherein step (c) is performed after step (b).

13. The method of any one of the preceding claims, further comprising, after step (b), adding a wash buffer to methanol treated cells and filtering cells through a sterile filter.

14. The method of any one of the preceding claims, wherein the methanol treated cells are incubated in the blocking buffer for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, overnight, 24 hours, or between 10 and 60 minutes.

15. The method of any one of the preceding claims, wherein the blocking buffer comprises:(i) a saline sodium citrate (SSC) buffer;(ii) a DNA blocking agent;(iii) a reducing agent; and(iv) an RNase inhibitor.

16. The method of claim 15, wherein the blocking buffer comprises:(i) about 0.1 to 30X SSC buffer;(ii) about 0.01% to 5% of Bovine Serum Albumin (BSA);(iii) about 0.01 pg / mL to 5000 pg / mL of the DNA blocking agent;(iv) about 0.1% to 20% of the reducing agent;(v) about 0.01% to 15% of the RNase inhibitor; and(vi) molecular grade / nuclease free water.

17. The method of either claim 15 or 16, wherein the SSC buffer comprises sodium chloride, sodium citrate, and / or trisodium citrate.

18. The method of any one of claims 15-17, wherein a formulation of IX SSC comprises a range of about 25 mM to 300 mM NaCl and a range of about 15 mM to 50 mM sodium citrate in a pH range of about 6.5 -7.5.

19. The method of claim 18, wherein the IX SSC buffer comprises about 150 mM NaCl and about 15 mM sodium citrate at a pH of about 7.0.

20. The method of either claim 18 or 19, wherein the SSC buffer is used at a 3X formulation.

21. The method of either claim 18 or 19, wherein the SSC buffer is used at a 5X formulation.

22. The method of any one of the preceding claims, further comprising, after incubating the methanol fixed cells in the blocking buffer, removing the blocking buffer; resuspending the methanol treated cells in the antibody reagent; and incubating the methanol treated cells in the antibody reagent.

23. The method of any one of the preceding claims, wherein the methanol treated cells are incubated in the antibody reagent at 0°C to room temperature.

24. The method of claim 23, wherein the methanol fixed cells are incubated in the antibody reagent for at least 5 minutes.

25. The method of claim 23 or 24, wherein the methanol fixed cells are incubated in the antibody reagent for at least 16 hours.

26. The method of any of the preceding claims, wherein the antibody reagent comprises:(i) 0. IX to 20X Phosphate buffered saline (PBS);(ii) 0.01 pg / mL to 5000 pg / mL of the DNA blocking agent;(iii) 0.01% to 20% bovine serum albumin;(iv) nuclease free water,(v) at least one antibody, and(vi) the RNase inhibitor.

27. The method of claim 26, wherein the antibody reagent comprises:(i) about 2-8% 20X PBS;(ii) about 50 pg / mL to 200 pg / mL the DNA blocking agent;(iii) about 0.4-5% bovine serum albumin;(iv) about 1-4% of the RNase inhibitor;(v) about 0.001 pg / mL to 100 pg / mL of at least one antibody; and(vi) nuclease free water.

28. The method of claim 27, wherein the antibody reagent comprises:(i) about 4-5% 20X PBS;(ii) about 100 pg / mL to 150 pg / mL of the DNA blocking agent;(iii) about 0.8-1.2% bovine serum albumin;(iv) about 2-3% of the RNase inhibitor;(v) about 0.025 pg / ml to 5 pg / ml of at least one antibody; and(vi) nuclease free water.

29. The method of any one of claims 26-28, wherein the at least one antibody comprises more than one antibody.

30. The method of any one of claims 26-29, wherein the at least one antibody is a conjugated antibody.

31. The method of claim 30, wherein the conjugated antibody is conjugated to a DNA sequence or RNA sequence.

32. The method of claim 31, wherein the DNA is single stranded DNA.

33. The method of claim 31 or 32, wherein the DNA is at least 10 nucleotides long.

34. The method of any one of claims 31 to 33, wherein the DNA ranges between 20 to 100 nucleotides in length.

35. The method of any one of claims 26-34, wherein the at least one antibody is an antibody to an individual target protein, a protein complex, a post-translational modification in a protein, and / or a protein / nucleic acid complex.

36. The method of claim 35, wherein the target proteins include peptides, enzymes, hormones, and structural components.

37. The method of any one of claims 26-36, wherein the at least one antibody is an antibody directed to: T-bet / TBX21, CD3 (UCHT1), CD8a (SKI), S100A9, CD4 (RPA-T4), TCF1 / TCF7, Ikaros, Aiolos, CD19, Ibal / AIF-1, Phospho-CREB, CREB, Phospho-Akt, Akt, phospho-p44 / 42 MAPK (Erkl / 2), p44 / 42 MAPK (Erkl / 2), Vimentin, phospho-stat3, stat3, phospho-stat4, stat4, phospho-statl, statl, NCAM1, Phospho-Histone H3, histone H3, acetyl-histone H3, tri-methyl- histone H3, GAPDH, Mouse (G3A1) mAb IgGl Isotype Control, Phospho-S6 Ribosomal Protein, S6 ribosomal protein, phospho-4E-BPl, CD68, CDl lb / ITGAM, IFI16, phosphor-Zap- 70, Zap-70, NF ATI, BATF, phospho-glucocorticoid receptor, glucocorticoid receptor, Phospho- PLCyl, PLCyl, Phospho-IRF-3, IRF-3, NF-KB p65, phospho-NF-KB p65, FoxOl, PLCyl, phospho-cJun, eJun, IL-17F, IL-17A, caspase-3, phospho-histone H2A.X, histone H2A.X, phospho-YAP, YAP, phospho-p38 MAPK, p38 MAPK, phospho-Rb, Rb (4H1), NRF2, phospho-PTEN, PTEN, PCNA, SMAD2 / 3, phospho- EGF receptor, EGF receptor, c-Myc / N- Myc, phospho-SMAD2, ATF-4, phospho-mTOR, mTOR, phospho-eIF2a, phospho-TBKl / NAK, eIF2a, TBK1 / NAK, Phospho- SAPK / JNK, SAPK / JNK, COL1A1, phospho-c-Fos, c-Fos, Phospho-AMPKa, AMPKa, phospho-Stat6, Stat6, or E-cadherin.

38. The method of any one of claims 26-37, wherein the at least one antibody is directed to a post-translational modification of a target protein.

39. The method of claim 38, wherein the post-translational modification is phosphorylation, methionine oxidation, deamidation, glycosylation, ubiquiti nation, carbamylation, S- carboxymethylation, acetylation, or methylation.

40. The method of any one of the preceding claims, wherein the method further comprises washing the sample with a wash buffer after step (b) and before step (c).

41. The method of claim 40, wherein the wash step after step (b) and before step (c) comprises washing the cells with a buffer comprising at least 0.5-10X PBS and / or 0.01-10% BSA; and the wash is optionally repeated at least once.

42. The method of any one of the preceding claims, wherein the method further comprises washing the sample with a wash buffer after step (c) and before step (d).

43. The method of claim 42, wherein the wash buffer is a modified wash buffer comprising lx PBS with 0.5% BSA; and optionally RNase inhibitor.

44. The method of claim 42 or 43, wherein the washing step after step (c) and before step (d) is repeated at least once.

45. The method of any one of the previous claims, further comprising counting the methanol treated cells.

46. The method of any one of claims 15-45, wherein the DNA blocking agent is free DNA from at least one source, free RNA from at least one source, a combination of free DNA from at least two sources, a combination of free RNA from at least two different sources, or a combination of free DNA from at least one source and free RNA from at least one source.

47. The method of claim 46, wherein the at least one source for the free DNA is salmon sperm.

48. The method of any one of the preceding claims, wherein the total RNAs in the cells are sequenced.

49. The method of any one of the preceding claims, wherein the cells present in single-cell form are captured using a high throughput single-cell method.

50. The method of claim 49, wherein the high throughput method utilizes microfluidics.

51. The method of any one of the preceding claims, further comprising after step (c), subjecting the cells to single cell sequencing analysis.

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