Methods and kits for single-cell transcriptome analysis and immune profiling

Abstract

The present disclosure relates generally to methods of uniquely labeling or barcoding molecules within immune cells such as B cells, or within nuclei derived therefrom. The present disclosure also relates to kits for uniquely labeling molecules within immune cells such as B cells, or within nuclei derived therefrom. In particular, the methods and kits may relate to the labeling of RNAs and / or cDNA encoding B cell receptors (BCRs) within such cells or nuclei.

Description

METHODS AND KIT'S FOR SINGLE-CELL TRANSCRIPTOME ANALYSIS AND IMMUNEPROFILIN GTECHNICAL FIELD

[0001] The present disclosure relates generally to methods of uniquely labeling or barcoding molecules within or originating from a nucleus or plurality of nuclei, a cell or plurality of cells, or one or more tissues, organs, or organisms, including for the labeling of RNAs and / or cDNAs within immune cells such as B cells in mammals such as humans or mice. The present disclosure also relates to improved workflows for single-cell sequencing protocols, e.g., for the cell-specific labeling of cells or nuclei, including for protocols involving low numbers of cells or nuclei.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present application claims the benefit of and priority to U.S. Provisional Application no. 63 / 614,344, filed December 22, 2023, U.S. Provisional Application no. 63 / 614,436, filed December 22, 2023, and U.S. Provisional Application no. 63 / 676,884, filed July 29, 2024, each of which is herein incorporated by reference in its entirety.BACKGROUND

[0003] Next Generation Sequencing (NGS) can be used to identify and / or quantify individual transcripts from a sample of cells. However, such techniques may be complicated to perform on individual cells in large samples. In such methods, RNA transcripts are generally purified from lysed cells (i.e., cells that have been broken apart), followed by conversion of the RNA transcripts into complementary DNA (cDNA) using reverse transcription. The cDNA sequences can then be sequenced using NGS. In such a procedure, all of the cDNA sequences are mixed together before sequencing, such that RNA expression is measured for a whole sample and individual sequences cannot be linked back to an individual cell.

[0004] Methods for uniquely labeling or barcoding transcripts from individual cells can involve the manual separation of individual cells into separate reaction vessels and can require specialized equipment. An alternative approach to sequencing individual transcripts in cells is to use microscopy to identify individual fluorescent bases. However, this technique can be difficult to implement and is limited to sequencing a low number of cells.

[0005] Single-cell sequencing can allow the identification of transcripts from individual cells. However, a limitation of single-cell RNA sequencing is the inability to detect every expressed gene in a given cell without exhaustive sequencing, which can be cost prohibitive due to the sequencing depth required per cell. This is problematic for applications such as immune profiling, where it is desired to detect specific immune sequences (such as genes encoding variable B-cell receptors, or BCRs) in every cell. For example, understanding the relationship between such naturally varying genes in populations of cells and the whole transcriptome in the same cells can provide critical insight into the biological and / or disease- related roles of each variant. For example, analyzing the relationship between B cell receptors (BCRs) and B cell activation during disease pathogenesis and progression, or during treatment with one or more therapeutic or candidate therapeutic agents, can assist, e.g., in the development of next-generation therapeutics with more favorable and sustainable outcomes. However, previous approaches to accomplishing this using, e.g., single-cell sequencing to profile receptor sequences and full transcriptomes, have been limited by a variety of factors, e.g., related to their requirement for microfluidics devices or plate-based protocols, and / or because of their limited sensitivity and throughput (l,000s-10,000s of cells), making these approaches both time consuming and costly.

[0006] B cells are immune cells that contribute to adaptive immunity by recognizing and eliminating a wide variety of immunologic threats, while maintaining self-tolerance. Their ability to recognize foreign antigens involves recognition of the antigens by the hypervariable B-cell receptors (BCR). B cell receptors each comprise four chains, including two light chains and two heavy chains. The heavy chains each comprise a transmembrane region, a constant region, and a variable region, while the light chains only provide a variable region. Each BCR comprises two antigen binding sites, with each site created by the variable regions of one heavy chain and one light chain. There are two types of BCR light chains, kappa and lambda, and numerous types of BCR heavy chains, including IgG, IgA, IgM, IgD, and IgE.

[0007] The hyper-variability in antigen recognition between different BCRs is produced in part by a process called V(D)J recombination, during which variable (V), diversity (D), and joining (J) segments are selected randomly from among a large number of possible segments in each cell to produce a vast repertoire of different segment combinations within the variable regions of each chain. The antigen-binding site in BCRs is made up of three variable “complementarity determining regions,” or CDRs, present in each chain making up thereceptor. Of these CDRs, CDR3 is particularly diverse, as it includes the V(D)J recombination junction, where random nucleotides may also be inserted during recombination.

[0008] In addition, pharmaceutical and other companies or organizations, as well as clinicians often work with low numbers of precious cell types, where avoiding cell loss during various analytical steps is of upmost importance. However, current methods for manipulating cells or nuclei for applications such as single cell sequencing, which require high final concentrations of fixed cells or nuclei for barcoding, are often limited by low cell retention, e.g., resulting from cells being lost during fixation, filtering, and / or centrifugation steps.

[0009] There is therefore a need for new methods for simultaneously assessing immune cell profiles in single cells, such as through determining BCR sequences together with the overall transcriptome, that are high-throughput, sensitive, and free of costly or cumbersome requirements for, e.g., microfluidics devices or plate-based protocols. In addition, there is a need for new and improved methods for handling cells and nuclei during steps of single-cell sequencing protocols such as fixation and / or barcoding steps, that allow for high rates of cell retention while maintaining a high level of quality of sequencing libraries and data. The present disclosure addresses these needs and provides other advantages as well.SUMMARY

[0010] The present disclosure provides methods, compositions, kits, and systems for labeling RNA and other molecules in and originating from immune cells such as B cells. The present methods relate to methods of labeling, in parallel, immune-specific molecules such as B-cell receptors (BCRs) and the transcriptomes of single cells, e.g., the whole transcriptome or a subset of the transcriptome.

[0011] In one aspect, the present disclosure provides a method of uniquely labeling RNA molecules within a plurality of B cells, the method comprising (a) fixing and permeabilizing a plurality of B cells; (b) dividing the plurality of B cells into a first plurality of aliquots, wherein each aliquot comprises more than one cell; (c) generating complementary DNA (cDNA ) molecules within the B cells of the first plurality of aliquots, wherein the RNA molecules are reverse transcribed using reverse transcription (RT) primers each comprising: (i) a poly(T) sequence or a random sequence; and (ii) an RT barcode sequence, wherein the RT barcode sequences present within the RT primers are specific to each aliquot; (d) pooling the B cells from the first plurality of aliquots; (e) tagging the cDNA molecules within the pooled B cells with one or more nucleic acid tags by performing steps (e)(i) through (e)(iii) one or more times:(i) dividing the pooled B cells into an additional plurality of aliquots; (ii) coupling nucleic acid tags to the cDNA molecules within the B cells of the additional plurality of aliquots, thereby generating a plurality of tagged cDNA molecules within each cell, wherein each nucleic acid tag comprises a tag barcode sequence, and wherein the tag barcode sequences present within the nucleic acid tags are specific to each aliquot; (iii) combining the B cells from the additional plurality of aliquots; (f) dividing the combined B into a plurality of sublibraries; (g) lysing the B cells in one or more sublibrary of the plurality of sublibraries to release the plurality of tagged cDNA molecules from each cell and produce a lysate comprising the released tagged cDNA molecules; (h) isolating the released tagged cDNA molecules from the lysate; (i) preparing separate sequencing libraries from the released tagged cDNA molecules for: (i) the whole transcriptome or a subset thereof, and (ii) BCR-encoding transcripts within the transcriptome.

[0012] In some embodiments of the method, preparing the sequencing libraries comprises amplifying the released tagged cDNA molecules during one or more rounds of amplification, wherein at least a portion of the primers used to amplify the released tagged cDNA molecules in one or more of the rounds of amplification comprise an index sequence, and wherein the index sequences present within the at least a portion of the primers are specific to each sublibrary. In some embodiments, preparing the sequencing library for the BCR-encoding transcripts comprises enriching the released tagged cDNA molecules for cDNA sequences encoding BCRs. In some embodiments, the released tagged cDNA molecules are enriched for cDNA sequences encoding BCRs by hybrid capture. In some embodiments, the hybrid capture is performed using probes targeting any one or more of the sequences shown in Table 13 and / or Table 14 and / or shown as SEQ ID NOS: 750-944 and / or 945-1121. In some embodiments, the hybrid capture is performed using probes targeting all of the sequences shown in Table 13 and / or all of the sequences shown in Table 14 and / or shown as SEQ ID NOS: 750-944 and / or 945-1121. In some embodiments, the hybrid capture is performed using probes targeting at least about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the sequences shown in Table 13 and / or Table 14 and / or shown as SEQ ID NOS: 750-944 and / or 945-1121. In some embodiments, the hybrid capture is performed using probes comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology or complementarity to at least about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequences shown in Table 13 and / or Table 14 and / or shown as SEQ ID NOS: 750-944 and / or 945-1121.

[0013] In some embodiments, the B cells comprise human B cells, and wherein the hybrid capture is performed using probes targeting any one or more of the sequences shown in Table13 and / or shown as SEQ ID NOS: 750-944, or targeting a subsequence of any of the sequences shown in Table 13 and / or shown as SEQ ID NOS: 750-944, or comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology or complementarity to at least about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequences shown in Table 13 and / or shown as SEQ ID NOS: 750-944. In some embodiments, the B cells comprise mouse B cells, and wherein the hybrid capture is performed using probes targeting sequences shown in Table 14 and / or targeting sequences shown as SEQ ID NOS: 945-1121, or targeting a subsequence of any of the sequences shown in Table 14 and / or shown as SEQ ID NOS: 945-1121, or comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology or complementarity to at least about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequences shown in Table 14 and / or shown as SEQ ID NOS: 945-1121. In some embodiments, the B cells comprise chimeric human-mouse B cells, and wherein the hybrid capture is performed using probes targeting sequences shown in Tables 13 and sequences shown in Table14 and / or targeting sequences shown as SEQ ID NOS: 750-944 and sequences shown as SEQ ID NOS: 945-1121, probes targeting any one or more of the sequences shown in Table 13 and any one or more of the sequences shown in Table 14 and / or targeting any one or more of SEQ ID NOS: 750-944 and any one or more of SEQ ID NOS: 945-1121, probes targeting a sequence or subsequence of any one or more of the sequences shown in Table 13 and targeting a sequence or subsequence of any one or more of the sequences shown in Table 14, and / or targeting a sequence or subsequence of any one or more of SEQ ID NOS: 750-944 and targeting a sequence or subsequence of any one or more of the SEQ ID NOS: 945-1121, and / or probes comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology or complementarity to at least about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequences shown in Table 13 and / or shown as SEQ ID NOS: 750-944, and comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology or complementarity to at least about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequences shown in Table 14 and / or shown as SEQ ID NOS: 945-1121.

[0014] In some embodiments, the hybrid capture is performed using one or more probes targeting one or more genes selected from the group IGHA1, IGHA2, IGHD, IGHD1-1,IGHD1-7, IGHD1-14, IGHD1-20, IGHD1-26, IGHD10R15-1A, IGHD10R15-1B, IGHD2-2, IGHD2-8, IGHD2-15, IGHD2-21, IGHD2OR15-2A, IGHD2OR15-2B, IGHD3-3, IGHD3-9, IGHD3-10, IGHD3-16, IGHD3-22, IGHD3OR15-3A, IGHD3OR15-3B, IGHD4-4, IGHD4- 11, IGHD4-17, IGHD4-23, IGHD4OR15-4A, IGHD4OR15-4B, IGHD5-5, IGHD5-12, IGHD5-18, IGHD5-24, IGHD5OR15-5A, IGHD5OR15-5B, IGHD6-6, IGHD6-13, IGHD6- 19, IGHD6-25, IGHD7-27, IGHE, IGHG1, IGHG2, IGHG3, IGHG4, IGHGP, IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, IGHJ6, IGHM, IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18, IGHV1- 24, IGHV1-38-4, IGHV1-45, IGHV1-46, IGHV1-58, IGHV1-69, IGHV1-69-2, IGHV1-69D, IGHV10R15-1, IGHV1OR15-5, IGHV1OR15-9, IGHV10R21-1, IGHV2-5, IGHV2-26, IGHV2-70, IGHV2-70D, IGHV2OR16-5, IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-16, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-30-3, IGHV3- 30-5, IGHV3-33, IGHV3-35, IGHV3-38, IGHV3-38-3, IGHV3-43, IGHV3-43D, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-64D, IGHV3-66, IGHV3-72, IGHV3-73, IGHV3- 74, IGHV3OR16-8, IGHV3OR16-9, IGHV3OR16-10, IGHV3OR16-12, IGHV3OR16-13, IGHV3OR16-17, IGHV4-4, IGHV4-28, IGHV4-30-1, IGHV4-30-2, IGHV4-30-4, IGHV4-31, IGHV4-34, IGHV4-38-2, IGHV4-39, IGHV4-59, IGHV4-61, IGHV4OR15-8, IGHV5-10-1, IGHV5-51, IGHV6-1, IGHV7-4-1, IGHV7-81, IGHV8-51-1, IGKC, IGKJ1, IGKJ2, IGKJ3, IGKJ4, IGKJ5, IGKV1-5, IGKV1-6, IGKV1-8, IGKV1-9, IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37, IGKV1-39, IGKV1D-8, IGKV1D-12, IGKV1D-13, IGKV1D-16, IGKV1D-17, IGKV1D-33, IGKV1D-37, IGKV1D-39, IGKV1D- 42, IGKV1D-43, IGKV1OR2-0, IGKV1OR2-108, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-40, IGKV2D-24, IGKV2D-26, IGKV2D-28, IGKV2D-29, IGKV2D-30, IGKV2D-40, IGKV3-7, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-7, IGKV3D-11, IGKV3D-15, IGKV3D-20, IGKV3OR2-268, IGKV4-1, IGKV5-2, IGKV6-21, IGKV6D-21, IGKV6D-41, IGLC1, IGLC2, IGLC3, IGLC6, IGLC7, IGLJ1, IGLJ2, IGLJ3, IGLJ4, IGLJ5, IGLJ6, IGLJ7, IGLV1-36, IGLV1-40, IGLV1-44, IGLV1-47, IGLV1-50, IGLV1-51, IGLV2- 8, IGLV2-11, IGLV2-14, IGLV2-18, IGLV2-23, IGLV2-33, IGLV3-1, IGLV3-9, IGLV3-10, IGLV3-12, IGLV3-16, IGLV3-19, IGLV3-21, IGLV3-22, IGLV3-25, IGLV3-27, IGLV3-32, and IGLV4-3.

[0015] In some embodiments, the hybrid capture is performed using probes that are from about 30-200 nucleotides long, or at least about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more nucleotides long, or about 50-150, 50-100, 100- 150, 100-120, 110-230, 120-140, or 130-150 nucleotides long. In some embodiments, thehybrid capture is performed using probes targeting from 50-500, 10-300, 50-100, 100-200, 200-300, 100-150, 150-200, 200-250, or about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 different regions within BCR transcripts. In some embodiments, the hybrid capture is performed using BCR-specific probes, and wherein at least a portion of the probes target transcripts encoding an IgM, IgG, IgD, IgA, and / or IgE isotype BCR. In some embodiments, the hybrid capture is performed using BCR-specific probes, and wherein at least a portion of the probes target transcripts encoding a BCR heavy chain. In some embodiments, the hybrid capture is performed using BCR-specific probes, and wherein at least a portion of the probes target transcripts encoding a BCR light chain. In some embodiments, the BCR light chains is a kappa and / or lambda light chain. In some embodiments, the hybrid capture is performed using BCR-specific probes, and wherein at least a portion of the probes target one or more constant regions of one or more BCR heavy or light chains. In some embodiments, the hybrid capture is performed using BCR-specific probes, and wherein at least a portion of the probes target one or more variable regions of one or more BCR heavy or light chains.

[0016] In some embodiments, the B cells comprise transitional B cells, naive B cells, memory B cells, plasma B cells, B cell progenitors, plasmablasts, plasma cells, lymphoplasmacytoid cells, follicular B cells, marginal-zone B cells, Bl cells, peripheral B cells, and / or regulatory B cells. In some embodiments, the B cells are isolated from the bone marrow, spleen, lymph nodes, blood, lymph, secondary lymphoid organs, appendix, intestine, Peyer’s patches, tonsils, thymus, and / or lymphoid follicles. In some embodiments, the B cells comprise activated B cells. In some embodiments, the B cells comprise primary B cells. In some embodiments, the B cells are derived from a cell line. In some embodiments, the B cells express any one or more of the genes selected from CD1, CD5, CD19, CD20, CD21, CD23, CD24, CD25, CD27, CD30, CD34, CD38, CD40, CD45R, CD78, CD80, C138, CD319, Notch2, TLR4, PD-L2, IL-6, IL-10, TGFb, CXCR3, CXCR4, CXCR5, CXCR6, IgM, IgG, IgA, IgE, and IgD.

[0017] In some embodiments, during at least one of the performances of steps (i) through (iii) each of the nucleic acid tags further comprises one or more elements selected from a capture agent, an additional sequence that allows or facilitates the detection of duplicates arising during PCR, and a NGS adapter sequence. In some embodiments, the hybrid capture is performed using a set of nucleic acid probes designed to capture the entire BCR repertoire present in the plurality of cells. In some embodiments, hybrid capture is performed using a setof nucleic acid probes comprising one or more probes specific for one or more of IgM, IgG, IgD, IgA, and IgE isotypes, IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2 subclasses, BCR heavy chain, BCR light chain, BCR heavy chain constant region, BCR heavy chain variable region, BCR light chain constant region, BCR light chain variable region, BCR mu heavy chain, BCR delta heavy chain, BCR gamma heavy chain, BCR epsilon heavy chain, BCR alpha heavy chain, BCR kappa light chain, BCR lambda light chain, BCR complementarity-determining region (CDR), BCR CDR1, BCR CDR2, BCR CDR3, BCR CDR-H1, BCR CDR-H2, or BCR CDR-H3 domain.

[0018] In some embodiments, the method further comprises grouping sequencing reads according to one or more features selected from the group consisting of RT barcode sequences, tag barcode sequences, series or combinations of tag barcode sequences, and index sequences. In some embodiments, the grouped sequencing reads are used to determine the individual B cell from among the plurality of B cells from which a given transcript originated. In some embodiments, the method further comprises grouping sequence reads into BCR gene sequences and non-BCR gene sequences. In some embodiments, the method further comprises relating the identity of the specific BCR gene sequences and / or heavy or light chain variable region sequences expressed in a given individual cell to the pattern of expression of non-BCR gene sequences in the same individual cell. In some embodiments, the method comprises identifying paired BCR heavy and light chain gene sequences within the same individual cell. In some embodiments, the method further comprises determining the relative abundance of one or more BCR clonotypes among the plurality of cells.

[0019] In some embodiments, the plurality of cells were all isolated from a single subject. In some embodiments, the plurality of cells comprise cells obtained at two or more different time points from the subject. In some embodiments, the method comprises determining the relative abundance of one or more BCR clonotypes among the plurality of the cells, and further comprising determining the evolution of the clonotypes between the two or more different time points. In some embodiments, the subject has a disease and / or has been administered a compound or other agent susceptible to affect the activity, presence, and / or role of B cells in the subject. In some embodiments, the whole transcriptome sequencing library and the BCR sequencing library are sequenced separately. In some embodiments, the whole transcriptome sequencing library and the BCR sequencing library are sequenced together. In some embodiments, one or more of steps (a), (b), (d), (e)(i), (e)(iii), or (f) are carried out at a temperature of below about 8, 7, 6, 5, 4, 3, 2, 1, 0, -1, -2, -3, or -4 °C, between about -4 to 8, -4 to 0, 0 to 4, 4 to 8, or 0 to 8 °C, or at about 8, 7, 6, 5, 4, 3, 2, 1, 0, -1, -2, -3, or -4 °C. In some embodiments, the method comprises generating second strands of the released tagged cDNA molecules to produce double-stranded cDNA molecules prior to step (h). In some embodiments, the second strands of the released cDNA molecules are generated by template switching. In some embodiments, the template switching introduces a common adapter sequence to the 3 ’-end of the released cDNA molecules. In some embodiments, the nucleic acid tags are coupled to the cDNA molecules in step (e)(ii) by ligation. In some embodiments, the RT primers each comprise a 5’ overhang comprising a 5’ overhang sequence. In some embodiments, the nucleic acid tags each comprise a first strand comprising a 3’ hybridization sequence and / or a 5’ hybridization sequence flanking the 3’ end and / or the 5’ end of the tag barcode sequence, respectively.

[0020] In some embodiments, the RT primers each comprise a 5’ overhang comprising a 5’ overhang sequence, and wherein the nucleic acid tags each further comprise a second strand comprising: a first portion complementary to a 5’ hybridization sequence of a previously coupled nucleic acid tag or a 5’ overhang sequence of an RT primer; and a second portion complementary to the 3’ hybridization sequence.

[0021] In some embodiments, one or more of the pluralities of aliquots or sublibraries are distributed in a 96-well plate. In some embodiments, one or more of the 96-well plates comprise unique dual indexes. In some embodiments, the additional sequence that allows or facilitates the detection of duplicates arising during PCR comprises random or degenerate bases. In some embodiments, the capture agent comprises biotin.

[0022] In another aspect, a kit is provided for performing any of the herein-disclosed methods. In some embodiments, the kit comprises one or more probe comprising at least about 70%, 75%, 80%, 85%, 90%, 96%, 97%, 98%, 99%, or 100% identity or complementarity to any one or more of the sequences shown in Table 13 or Table 14 or shown as SEQ ID NOS: 750-944, or a subsequence thereof. In some embodiments, the subsequence comprises at least about 30, 40, 50, 60, 70, 80, 90, 100, or more nucleotides.

[0023] In another aspect, the present disclosure provides a method for preparing cells or nuclei for single-cell sequencing, the method comprising: (a) providing a plurality of cells or nuclei distributed in one or more wells or containers, wherein the plurality of cells or nuclei in the one or more wells or containers are suspended in a prefixation buffer; (b) fixing the plurality of cells or nuclei by adding a cell or nucleus fixation solution to the one or more wells orcontainers comprising the plurality of cells or nuclei; (c) permeabilizing the plurality of cells or nuclei by adding a cell or nucleus permeabilization solution to the one or more wells or containers comprising the plurality of cells or nuclei; (d) adding a neutralization buffer to the one or more wells or containers comprising the plurality of cells or nuclei, such that the neutralization buffer prevents further fixation and / or permeabilization of the cells or nuclei; (e) freezing the plurality of cells or nuclei; (f) thawing the plurality of cells or nuclei; (g) adding cell binding beads to the plurality of thawed cells or nuclei such that the cell binding beads bind to the plurality of thawed cells or nuclei; (h) placing the one or more wells or containers on a magnet, such that the cells or nuclei bound to the cell binding beads localize to a location within each of the wells or containers in proximity to the magnet; (i) removing the supernatant from the one or more wells or containers comprising the localized plurality of cells or nuclei bound to the cell binding beads; and (j) removing the one or more wells or containers from the magnet and resuspending the plurality of cells or nuclei in the one or more wells or containers in a storage buffer.

[0024] In some embodiments, the supernatant is removed from the one or more wells or containers by pipetting. In some embodiments, the cell binding beads are coated with concanavalin A (ConA). In some embodiments, the method further comprises counting the plurality of cells or nuclei in one or more of the wells or containers prior to step (b) and subsequent to step (j), wherein the number of cells or nuclei counted subsequent to step (j) is at least 70%, 75%, 80%, 85%, 90%, or 95% of the number of cells counted prior to step (b). In some embodiments, the beads are added at a ratio of beads to cells / nuclei of 4 L beads for 10,000-49,999 cells or nuclei, 6 L beads for 50,000-74,999 cells or nuclei, 9 L beads for 75,000-89,999 cells or nuclei, or 12 L beads for 90,000-100,000 cells or nuclei.

[0025] In some embodiments, the cells or nuclei are any one of more of mammalian cells or nuclei, human cells or nuclei, mouse cells or nuclei, primary cells, cell lines, PBMCs, cells isolated from a tissue, HEK cells, 3T3 cells, T cells, B cells, or nuclei isolated from primary cells, cell lines, PBMCs, cells isolated from a tissue, T cells, B cells, HEK cells, 3T3 cells, or fungal cells. In some embodiments, subsequent to step (j) the cells or nuclei are used in a single cell sequencing method comprising sequencing RNA and / or genomic DNA. In some embodiments, the plurality of cells is distributed in a multi-well plate. In some embodiments, the multi-well plate is a 96-well plate.

[0026] In another aspect, the present disclosure provides a method of preparing cells or nuclei for single-cell sequencing, the method comprising: (a) providing a plurality of cells or nuclei distributed in one or more wells or containers, wherein the plurality of cells or nuclei in the one or more wells or containers are suspended in a prefixation buffer; (b) fixing and permeabilizing the plurality of cells or nuclei by adding a cell or nucleus fixation solution and a permeabilization solution to the one or more wells or containers comprising the plurality of cells or nuclei; (c) adding a neutralization buffer to the one or more wells or containers comprising the plurality of cells or nuclei, such that the neutralization buffer prevents further fixation of the cells or nuclei; (d) freezing the plurality of cells or nuclei; (e) thawing the plurality of cells or nuclei; (f) adding a plurality of magnetic beads to the one or more wells or containers comprising the plurality of thawed cells or nuclei, wherein the plurality of magnetic beads are coated with an agent having affinity to molecules on the surface of the cells or nuclei such that the magnetic beads bind to the surface of the plurality of thawed cells or nuclei; (g) contacting the one or more wells or containers with a magnet such that the cells or nuclei bound to the magnetic beads become localized adjacent to the site of the magnet on the outside of each well or container; (h) removing the supernatant from the one or more wells or containers comprising the thawed plurality of cells or nuclei; and (i) resuspending the thawed plurality of cells or nuclei in the one or more wells or containers in a storage buffer.

[0027] In some embodiments, the method further comprises performing a single-cell RNA sequencing method on the resuspended cells or nuclei. In some embodiments, the method further comprises performing a single-cell genomic DNA sequencing method on the resuspended cells or nuclei. In some embodiments, the beads are ConA beads. In some embodiments, the plurality of cells or nuclei comprises from about 10,000 to about 100,000 cells or nuclei. In some embodiments, wherein from about 4 L to about 12 L of beads are added to the plurality of cells or nuclei.

[0028] In some embodiments, the plurality of cells comprises from about 10,000 cells or nuclei to about 49,999 cells or nuclei, and about 4 L of beads are added. In some embodiments, the plurality of cells comprises from about 50,000 cells or nuclei to about 74,999 cells or nuclei, and about 6 L of beads are added. In some embodiments, the plurality of cells comprises from about 75,000 cells or nuclei to about 89,999 cells or nuclei, and about 9 L of beads are added. In some embodiments, the plurality of cells comprises from about 90,000 to 100,000 cells or nuclei, and about 12 L of beads are added. In some embodiments, the average cell retention rate during the method is at least about 75%, 80%, 85% 90%, or 95%. Insome embodiments, the doublet or multiplet rate during the method is less than about 15%, 10%, or 5%.

[0029] In some aspects, implementations of any of the present methods include hardware, e.g., a device, apparatus, or system configured to perform any of the herein-disclosed methods or processes, and / or computer software on a computer-accessible medium.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 provides an overview of one embodiment of the present method. In this embodiment, cells or nuclei are fixed and permeabilized, cDNA is generated within the cells or nuclei by reverse transcription, e.g., using well-specific barcoded primers, one or more additional barcodes are appended to the cDNA within the cells or nuclei by a split-pool labeling process to couple cell-or nucleus specific barcode sequences (or barcode sequence combinations) to each cDNA, the cells or nuclei are lysed, the cDNA is amplified, and the amplified cDNA molecules are used to prepare, in parallel, a Whole Transcriptome sequencing library and a BCR sequencing library (involving, e.g., enrichment of BCR sequences using hybrid capture as described herein). Finally, the sequencing libraries are sequenced, and the sequencing reads analyzed.

[0031] FIGS. 2A-2D provide an overview of in situ cell barcoding steps (i.e., combinatorial barcoding, or split-pool labeling) performed in various embodiments of the present methods. FIG. 2A: Round 1 Barcoding. Fixed and permeabilized cells are loaded into a multiple wells (e.g., 48 wells) of a Round 1 plate. RNA is reverse transcribed to generate cDNA using reverse transcription primers comprising a well-specific barcode (“BC1”) and, e.g., a poly(dT) sequence or a random sequence. FIG. 2B: Round 2 Barcoding: The cells containing the cDNA are pooled and loaded into a Round 2 Plate. An adapter (nucleic acid tag) with a well-specific barcode (“BC2”) is ligated to the cDNA, e.g., 5’ to the first barcode. FIG. 2C: Round 3 Barcoding: The cells are pooled and loaded into a Round 3 Plate. A third barcode is ligated to the cDNA (indicated in red) via another adapter, which also contains an Illumina R2 sequence, and biotin. FIG. 2D: Lysis and Sublibrary Generation: Cells are split into multiple sublibraries (or “samples”) (e.g., 8 sublibraries or samples) and lysed.

[0032] FIGS. 3A-3C provide an overview of cDNA capture and amplification steps performed in various embodiments of the present methods. FIG. 3A: cDNA Capture: Following cell lysis, biotinylated cDNA is captured (isolated) in each sublibrary via streptavidin beads. FIG. 3B: cDNA Template Switch: A template switch (TS) reaction adds anadapter to the 3’ end of the cDNA. FIG. 3C: cDNA Amplification: the cDNA is amplified by PCR using primers binding, e.g., to a Template Switch (TS) sequence and to an NGS adapter sequence such as the Illumina TruSeq R2 sequence.

[0033] FIGS. 4A-4C show steps in the preparation of sequencing libraries starting from amplified cDNA molecules as shown, e.g., in FIG. 3C. FIG. 4A: the cDNA molecules are fragmented, and the ends are repaired and then A-tailed. FIG. 4B : Adapter Ligation: An adapter such as an Illumina TruSeq R1 Adapter is ligated to the 5’ end of the DNA. FIG. 4C: Round 4 Barcoding: The sequencing library is amplified, adding, e.g., P5 / P7 Adapters and a fourth barcode via the UDI - WT Plate.

[0034] FIGS. 5A-5B show steps in the preparation of BCR sequencing libraries starting from amplified cDNA molecules as shown, e.g., in FIG. 3C. FIG. 5A: WT Libraries Pool and Blocker Hybridization: Up to 8 WT sublibraries are pooled per BCR capture reaction. Evercode library-specific and repetitive sequences blockers are hybridized. FIG. 5B: BCR Hybridization: Biotinylated probes in the Human BCR Panel are hybridized to the target DNA.

[0035] FIGS. 6A-6B show steps in the amplification of BCR cDNAs hybridized by probes during hybridization, e.g., as shown in FIG. 6A. Bind and wash: Target molecules are captured with Streptavidin Binder Beads and washed. FIG. 6B: BCR Enrichment: Captured molecules are enriched via PCR. The amplified double stranded libraries are ready to sequence.

[0036] FIG. 7: Whole transcriptome and BCR sequencing library structure. The diagram illustrates the composition of the sequencing libraries generated. This library structure applies to both the whole transcriptome library and the BCR-enriched library.

[0037] FIGS. 8A-8C illustrate exemplary nucleic acid tags and their use according to certain embodiments of the present disclosure. FIG. 8A depicts a nucleic acid tag, e.g., a Round 2 (R2) nucleic acid tag, with the first and second strands preannealed. The first strand is shown below, and the second strand is shown above. The annealed first and second strands of the nucleic acid tag comprise a central duplex region comprising the annealed first and second strand barcode sequences, and the annealed second strand hybridization sequence and first strand 3’ hybridization sequence. The depicted tag has an overhang at either end of the central duplex, with one overhang comprising the first strand 5’ hybridization region, and the other overhang comprising the second strand overhang sequence. The relative lengths of the sequences in the illustration are not drawn to scale. FIG. 8B shows the nucleic acid tag fromFIG. 8 A coupled to a barcoded (Rl) reverse transcription (RT) primer. The second strand overhang from the nucleic acid tag of FIG. 8 A is annealed to the 5’ overhang sequence of the RT primer. FIG. 8C shows the nucleic acid tag from FIG. 8A coupled to another nucleic acid tag, e.g., a nucleic acid tag coupled to a cDNA molecule or genomic DNA fragment in a previous round of tagging. The second strand overhang from the nucleic acid tag of FIG. 8A is annealed to the 5’ hybridization sequence of the second nucleic acid tag.

[0038] FIGS. 9A-9C provide examples of various types of fixed cells following staining with trypan blue. FIG. 9A: high quality fixed samples have single distinct cells with <5% cell aggregation and no debris. FIG. 9B: some aggregation of cells is present. FIG. 9C: cell debris is present; when quantifying fixed cells, it is critical to avoid counting such debris, which could lead to overestimations of the number of cells.

[0039] FIGS. 10A-10B. Expected post-amplification sublibrary cDNA size distribution. Example trace of Human cDNA (FIG. 10A) and Mouse cDNA (FIG. 10B) run on a Tapestation.

[0040] FIGS. 11A-11B. Sequencing Library Quantification. Expected Size Distribution of cDNA libraries before Illumina Sequencing (e.g., following section 3.6 in Example 1). Example trace of Human DNA (FIG. 11 A) and Mouse DNA (FIG. 11B) from indexed sublibraries run on a TapeStation.

[0041] FIGS. 12A-12B. BCR library quantification. Expected Size Distribution of BCR libraries before Illumina Sequencing (e.g., following section 5.2 in Example 1). Example trace of Human DNA (FIG. 12A) and mouse DNA (FIG. 12B) from indexed sublibraries run on a TapeStation.

[0042] FIGS. 13A-13B. Fixation of cells and nuclei. FIG. 13A: Cells in suspension are fixed and permeabilized before undergoing the split-pool combinatorial barcoding steps. FIG. 13B: Nuclei in suspension are fixed and permeabilized before undergoing the split-pool combinatorial barcoding steps.

[0043] FIG. 14. A cell fixation workflow for 100,000 to 1 million cells, e.g., for BCR profiling.

[0044] FIG. 15. Exemplary Centrifugation Optimization Experiment. In this example, the sample is first centrifuged at 200 x g for 10 minutes. The pellet is then resuspended in Cell Storage Master Mix, and the first supernatant is centrifuged again at 300 x g for 10 minutes.The second pellet is resuspended in Cell Storage Master Mix, and the second supernatant is centrifuged again at 400 x g for 10 minutes. This final, third pellet is resuspended in Cell Storage Master Mix and the third supernatant is discarded. The three resuspended pellets can then be counted, e.g., using a hemocytometer. In the example shown in FIG. 15, the cells centrifuged at 400 x g are aggregated with significant debris, indicating that this resuspended pellet should be discarded. Conversely, the cells centrifuged at 200 x g and 300 x g were of high quality, so they were pooled together, allowing a calculation of~50% retention for this sample. These results suggest that this sample type should be centrifuged at 300 x g.

[0045] FIG. 16. A high throughput (e.g., for 1-4 million cells) cell fixation workflow, e.g., for BCR profiling.

[0046] FIG. 17. A low Input Cell / Nuclei Fixation workflow, designed for 12 (e.g., in tubes) or 96 reactions (e.g., in plates). In the first step, cells or nuclei present in 20 mL of buffer or media are centrifuged, and the supernatant is removed. In the next step, 60 mL of prefixation buffer is added, and the cells or nuclei are strained. Next, 25 mL of cell / nuclei fixation master mix is added, and the cells or nuclei are incubated on ice for 10 minutes. Next, 8 mL of permeabilization solution is added, and the cells or nuclei are incubated on ice for 3 minutes. Next, 110 mL of fix and perm stop is added. The cells or nuclei can then be frozen, e.g., at -80 °C. At a later time, the tubes or plates are thawed, and 4-12 mL of cell-binding beads (e.g., ConA beads) are added to each tube or well. The cells / nuclei and beads are mixed 3 times. Next, the tubes or plates are placed on a magnet for 5 minutes, and the supernatant is then removed. The cells / nuclei are then resuspended in cell / nuclei storage buffer, and can then be used in any of a variety of single-cell labeling protocols such as Evercode combinatorial barcoding, immune profiling (e.g., BCR or TCR profiling), or others.

[0047] FIGS 18A-18B provide examples of post-bind AO / DAPI stained HEK cells (FIG. 18 A) and PBMCs (FIG. 18B).

[0048] FIGS. 19A-19B: 24 samples and 1 million cells were analyzed in a single experiment. FIG. 19A: unsupervised clustering and annotation using whole transcriptome data found expected B cell subsets and other immune cells. FIG. 19B: Cell distribution across 24 samples, colored by donor, highlights the relative even distribution of B cells subtypes across all samples.

[0049] FIG. 20: Canonical marker genes were useful for cell identification.

[0050] FIG. 21 : 900,000+ clones and sensitive CDR3 detection allowed high BCR Chain Detection Across healthy and diseased samples. High rates of chain assignments resulted in over IM CDR3s were detected. Heavy chain CDR3 detection varied from 85-96%, paired CDR3 detection varied between 54-89%, and light chain CDR3 detection from 69-95%.

[0051] FIG. 22 shows the number of Unique vs. Expanded Clones split by Donor. Total unique clones varied between 13,000 - 59,000 per donor where all donors had over 95% unique clones in their clone pool with donor H-l and T1D-5 having the most expanded clonotypes at near 5%. A clonotype was defined as a cell having exact matched and paired CDR3s.

[0052] FIGS. 23A-23B. FIG. 23A: BCR chain use reveals biological complexity. T1D BCR Heavy Chain Use Split by Donor. Each line represents a unique clonotype where the width of the line is scaled to the number of cells per clonotype. Cells were selected for clones with >5 cells per clone. Both donor T1D-3 and T1D-4 had few expanded clones whereas Interestingly, donor T1D-5 was found to have a clear and robust IGHA BCR response suggesting that this specific patient had undergone an immune response. The majority of class switched memory B cells are IgG positive where the unswitched cells are IGHM. FIG. 23B: Variable V-gene use reveals bias toward IGHV3-23.

[0053] FIGS. 24A-24B: CDR3 diversity spans the full heavy chain, and CDR diversity increases from CDR1 to CDR3. FIG. 24 A: The CDR3 region had a median length of 17 amino acids where the CDR1 and CDR2 regions were between 7-8 amino acids long. FIG. 24B: Expanded clonotypes stand out amid the diversity of the CDR3 region.

[0054] FIG. 25 shows the retention of PBMCs (50,000 / sample) with three different volumes (5 mL, 5.5 mL, and 6 mL) of ConA beads.

[0055] FIGS. 26A-26C provide overviews of various fixation workflows provided by the present disclosure. In FIG. 26A, magnetic beads having affinity to the cell or nuclear surface such as ConA beads are added to cells or nuclei following fixation, permeabilization, and neutralization steps. In FIG. 26B, beads such as ConA beads are added before fixation. In FIG. 26C, beads are added after fixation, permeabilization, neutralization, and freezing of the cells or nuclei (in DMSO), with the beads then being added after thawing of the cells or nuclei.

[0056] FIG. 27 provides an overview of several alternative fixation workflows provided by the present disclosure, e.g., as illustrated in FIGS. 26A-26C. FIG. 27 illustrates several different points at which beads can be added. Each point offers certain potential advantages.For example, for point 1 (i.e., beads added before fix): this would eliminate all subsequent centrifugation steps. For points 2 or 3 (beads added during neutralization): this would cut out one fix centrifugation step, and would allow concentration of the samples before loading R1 plates. For point 6 (beads added after thaw): beads can be used for concentrating samples and can also be used for subsequent magnet-based split-pool labeling workflow. Also, point 6 avoids freezing and thawing beads, which may negatively affect subsequent steps.

[0057] FIGS. 28A-28E provide results indicating that higher concentrations of ConA beads are associated with reduced cell clumping. FIG. 28A shows cells prebinding; FIG. 28B shows cells with 5% beads, at 4,500 cells / ml; FIG. 28C shows cells with 5% beads, at a final concentration of 1,500 cells / ml; FIG. 28D shows cells with 2.5% beads, at a final concentration of 1,500 cells / ml; FIG. 28E shows cells with two tested bead concentrations of below 2.5% (1% and 0.5%), resulting in significant clumping.

[0058] FIGS. 29A-29B provide results indicating that low concentrations of ConA beads (e.g., 1% by volume) do not adversely impact barcoding. FIG. 29A: Number of cells and multiplets detected with and without beads. FIG. 29B: UMAP showing clustering of the cells, with normal separation of the two cell types and with cells present in each cluster from both experimental conditions.DETAILED DESCRIPTIONIntroduction

[0059] The present disclosure relates generally to methods of uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and / or one or more tissues, organs, organisms, or subjects, and in particular to immune cells such as B cells (including individual B cells, a plurality of B cells, B cells within a tissue, B cells within an organ, B cell nuclei, etc.). The present disclosure also relates to compositions and kits for uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and / or a tissue, organ or organism, and in particular to immune cells such as B cells (including individual B cells, a plurality of B cells, B cells within a tissue, organ, or organism, B cell nuclei, etc.). The molecules to be labeled may include, but are not limited to, RNA molecules, cDNA molecules, DNA molecules, proteins, peptides, and / or antigens.

[0060] In various embodiments, the present disclosure provides methods and compositions for creating multiple related sequencing libraries, e.g., transcriptome sequencinglibraries for multiplex analyses. For example, in some embodiments, single cell whole transcriptome libraries are created that are coupled with BCR-specific libraries created using cDNA from the same cells. By creating such libraries, individual genes (such as genes encoding BCR chains) can be robustly detected across cells while limiting the overall sequencing needed, even when the individual genes are rare among the cells.

[0061] The present methods and compositions enable the enrichment and highly sensitive detection of individual (or a small number of, e.g., up to 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or more) target transcripts of interest in parallel with the whole transcriptome (or a subset thereof) in single cells. The target transcripts can correspond to any sequence of interest whose presence or expression level in a cell may be associated with other properties of interest of the cell. In particular embodiments, the target transcripts are BCR transcripts, i.e., a transcript encoding any one or more component of a B cell receptor (e.g., BCR heavy chain or BCR light chain).

[0062] In some embodiments, the analyses performed using the presently described methods can allow the detection or characterization of a disease process (e.g., the detection and / or tracking of B cell clonotypes) or the development, preparation, or monitoring of cell therapies (e.g., therapies involving modified B cells or BCRs).

[0063] It will be readily understood that the embodiments, as generally described herein, are exemplary. The following more detailed description of various embodiments is not intended to limit the scope of the present disclosure, but is merely representative of various embodiments. Moreover, the order of the steps or actions of the methods disclosed herein may be changed by those skilled in the art without departing from the scope of the present disclosure. In other words, unless a specific order of steps or actions is required for proper operation of the embodiment, the order or use of specific steps or actions may be modified.Definitions

[0064] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of, or consist of its particular stated element, step, ingredient, or component. As used herein, the transition term “comprise” or “comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of’ excludes any element, step, ingredient or component not specified. The transition phrase“consisting essentially of’ limits the scope of the embodiment to the specified elements, steps, ingredients or components, and to those that do not materially affect the embodiment.

[0065] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e., denoting somewhat more or somewhat less than the stated value or range, to within a range of, e.g., ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0066] The terms “a,” “an,” “the” and similar referents used in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0067] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0068] Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0069] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the following examples or when application of the meaning renders any construction meaningless or essentially meaningless in cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

[0070] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi -stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms “polynucleotide” and “nucleic acid” should be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides. Unless specifically limited, the term encompasses nucleic acids containing known analogs or derivatives of natural nucleotides, e.g., molecules that have similar binding properties as the reference nucleic acid. In some embodiments, the nucleic acids can comprise one or more modified nucleotides, e.g., nucleic acids modified at the base moiety, at the sugar moiety, or at the phosphate backbone (e.g.,phosphorothioates). In some embodiments, the nucleic acids can comprise one or more moieties to allow or facilitate, e.g., detection, quantification, purification, capture, identification, or selective removal, e.g., biotin, fluorescent labels, etc.

[0071] The term “gene” refers to the segment of DNA involved in producing a polypeptide chain or a non-coding transcript (e.g., mRNA). For coding sequences, it may include regions preceding and following the coding region (leader sequence and / or trailer sequence) as well as intervening sequences (introns) between individual coding segments (exons). A “transgene” refers to a gene that has been introduced into a cell or organism from another source (e.g., from another organism or following synthesis).

[0072] The terms “hybridizable” or “complementary” or “substantially complementary” it is meant that a nucleic acid (e.g. RNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e. form Watson-Crick base pairs and / or G / U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. As is known in the art, standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) [DNA, RNA], In addition, it is also known in the art that for hybridization between two RNA molecules (e.g., dsRNA), guanine (G) base pairs with uracil (U). For example, G / U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. In the context of this disclosure, a guanine (G) of a proteinbinding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule is considered complementary to a uracil (U), and vice versa. As such, when a G / U base-pair can be made at a given nucleotide position a protein-binding segment (dsRNA duplex) of a subject DNA- targeting RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary. As used herein, the terms “hybridize” or “complementary” refer to a first nucleotide sequence capable of forming non-covalently bind (hydrogen bond) with at least a portion of a specified second nucleotide sequence.

[0073] A "promoter" refers to a set of nucleic acid sequences that direct the transcription of a nucleic acid, e.g., an adjacent coding sequence. Promoters can be constitutive or inducible. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. Promotersas used herein can include bacterial promoters or eukaryotic promoters including RNA polymerase II (e.g., EF-1 alpha) and RNA polymerase III (e.g., U6) promoters. A promoter can also include distal enhancer or repressor elements. The promoter can be a heterologous promoter (i.e., not naturally linked to the coding sequence) or homologous (i.e., the promoter that naturally drives the expression of the transcribed sequence).

[0074] The term “binding” or “coupling” is used broadly throughout this disclosure to refer to any form of attaching or coupling two or more components, entities, or objects. For example, two or more components may be bound to each other via chemical bonds, covalent bonds, non-covalent bonds, ionic bonds, hydrogen bonds, electrostatic forces, Watson-Crick hybridization, nucleic acid sequence complementarity, etc.

[0075] An “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed (e.g., a protein coding sequence or a non-coding RNA such as a guide RNA), operably linked to a promoter. The promoter can be a heterologous promoter, i.e., a promoter not naturally linked to the transcribed sequence.

[0076] A “barcode” or “index” refers to a nucleotide sequence (the “barcode sequence” or “index sequence”) that is used to label an entity such as a cell, plurality of cells, cell populations, cell compartments, nucleic acids, polypeptides, or other molecules, and that varies among or between cells, cell populations, nucleic acids or other molecules, etc. For example, in some embodiments, a barcode is used to label (or tag) cDNAs generated within a given aliquot of cells, e.g., where all of the cDNAs labeled in the aliquot receive the same barcode, or receive a set of barcodes that is specific to the aliquot, i.e., that the specific set of barcodes used in the aliquot is different from the sets used in the other aliquots. Barcodes can be added to polynucleotides (or other molecules) in any of a number of ways. For polynucleotides, for example, they can be introduced, e.g., in a primer, template, template-switch oligonucleotide (TSO), or other polynucleotide used during a polymerization-based reaction such as reverse transcription, PCR, or other polymerization-based and / or amplification reaction; barcodes can also be added to polynucleotides by hybridization and / or by ligation, e.g., by ligation of an adaptor or other polynucleotide (e.g., a “nucleic acid tag”) comprising a barcode. Such adaptors or other barcode-comprising polynucleotides can be appended to a polynucleotide, e.g., byligation via blunt-end ligation, ligation to compatible restriction ends, ligation to A-tailed or otherwise tailed ends, using a linker strand, etc. The barcode, or adapter or other polynucleotide comprising the barcode, can be single-stranded, double-stranded, partially double-stranded and partially single-stranded (e.g., comprising one or more overhangs at the 3’ and / or 5’ ends), etc.

[0077] As used herein, when a polynucleotide is said to comprise a “barcode” (or the equivalent term “barcode sequence”) it means that the polynucleotide comprises a sequence of nucleotides that can be used to distinguish the polynucleotide comprising the barcode from one or more other polynucleotides, e.g., from polynucleotides originating from another cell, from polynucleotides labeled in a different aliquot or well, or from all other polynucleotides in a sample. In some embodiments, the barcode alone is sufficient to distinguish the polynucleotide from other polynucleotides, whereas in other embodiments the barcode provides information that can contribute to distinguishing the polynucleotide from other polynucleotides, but is not sufficient on its own (e.g., one or more additional sequence elements, or other markers, are also needed to completely distinguish the polynucleotide).

[0078] It will be appreciated that a “barcode” can refer to a single sequence of contiguous nucleotides, or to a combination of individual sequences of contiguous nucleotides. For example, in certain split-pool labeling methods as described in more detail elsewhere herein, multiple rounds of tagging can be performed, e.g., multiple rounds in each of which cells are divided into aliquots, nucleic acid tags (comprising a barcode) are added to molecules (such as cDNAs) in the cells of each aliquot, and the cells are then recombined (or repooled). As such, the tagged cDNA s in the cells of the aliquots can comprise two nucleic acid tags, each comprising a barcode. In some embodiments, the two barcodes present on the same molecule can be referred to herein as a single “barcode,” even if there are other sequence elements (such as linker sequences, adapter sequences, primer-binding sequences, etc.) intervening between the two barcodes on the molecule.

[0079] Further, it will be appreciated that when a polynucleotide is said to comprise a “barcode,” this can mean that, depending on the context, the specific sequence of the barcode (or “barcode sequence”) can vary between the different polynucleotides comprising the barcode, or that the specific barcode sequence is the same between the different polynucleotides. For example, in some embodiments, all of the cDNA s in cells of a given aliquot (or sample) are tagged with nucleic acid tags comprising the same barcode sequence, whereas the cDNAs of cells in other aliquots are tagged with nucleic acid tags comprising otherbarcode sequences. In some embodiments, however, a polynucleotide comprising a barcode is added to molecules in a given aliquot (or sample), wherein the specific barcode sequence differs between the different polynucleotides used in the aliquot or sample; such polynucleotides and barcodes can be used, for example, to distinguish between the different original molecules in the sample (e.g., to be able to detect errors arising during amplification of the original molecules, such as cDNAs derived from original mRNA molecules).

[0080] In some embodiments of the present disclosure, one or more barcodes is included in primers used for reverse transcription (i.e., RT primers) or amplification (e.g., PCR), or in a nucleic acid tag appended to a polynucleotide, e.g., by ligation, where the cells are divided into two or more (e.g., 2, 4, 8, 12, 16, 24, 32, 48, 96, or more) aliquots or wells prior to the reverse transcription, amplification, or ligation, and the barcode sequences used in the cells are aliquot- or well-specific. When barcode or index sequences are said to be “aliquot-specific” or “wellspecific,” this means that there is an association between the sequences used and the presence of the different cells within the two or more aliquots or wells, such that the association can be used to derive information about the location of a given cell within the aliquots or wells based upon the specific sequence. For example, in some embodiments each cell within a given aliquot or well has primers or tags with the same barcode sequence, and the barcode sequences are different between each aliquot or well. However, it will be appreciated that even where a less direct relationship exists between the barcode sequences and the aliquots or wells (e.g., where more than one barcode sequences are used within a given aliquot or well, or where more than one aliquot or well share one or more barcode sequences), the barcodes are still considered aliquot-specific or well-specific, so long that some information can be derived from the barcode sequence about the aliquot or well in which a given cell or nucleus was present.

[0081] “Split-pool labeling” or “split-pool barcoding” or “combinatorial labeling” or “combinatorial barcoding” refers to a cell-specific labeling method involving the use of fixed and permeabilized cells or nuclei as containers, wherein a plurality of the cells or nuclei are first separated into multiple wells (or aliquots), followed by the labeling of RNA or other molecules within each cell or nucleus using a well-specific tag or barcode, followed by the pooling of the cells, and wherein this cycle of separation, tagging, and pooling is repeated one or more times. In this way, at the end of the process each cell or nucleus within the plurality will comprise a combination of tags or barcodes that will reflect the particular combination of wells or aliquots in which it was present throughout the multiple rounds of tagging. As the number of rounds of tagging and / or the number of wells or aliquots used in each round isincreased, the number of potential barcode combinations increases correspondingly. As such, for a given number of cells or nuclei in the plurality a suitable experimental design can be prepared that will generate a high likelihood that tagged molecules, e.g., cDNAs, within each cell or nucleus will have the same combination of barcodes that is unique among the overall population of cells or nuclei. Examples of split-pool labeling methods are disclosed, e.g., in US Patent Nos. 10,900,065, 11,634,751, 11,168,355, 11,427,856, 11,555,216, 11,639,519, 11,680,283, 10,633,648, 11,421,221, US Pat. App. Pub. No. US 2021 / 0388415 Al, in Rosenberg et al., Science 360, 176-182 (2018), Rosenberg et al., BioRxiv (2017), “Scaling single cell transcriptomics through split pool barcoding,” doi.org / 10.1101 / 105163, Tran et al. BioRxiv (2022) “High sensitivity single cell RNA sequencing with split pool barcoding,” doi.org / 10.1101 / 2022.08.27.505512, the entire disclosures of all of which are herein incorporated by reference (including all supplemental material).

[0082] As used herein, the term “tagged cDNA molecules” refers to complementary DNA (cDNA) molecules comprising one or more barcodes (e.g., well-specific barcodes, cell-specific barcodes, or nucleus-specific barcodes), e.g., cDNA molecules generated within cells or nuclei to which a nucleic acid tag has been appended (e.g., by ligation). In some embodiments, the tagged cDNA molecules are tagged cDNA molecules corresponding to BCR transcripts, e.g., a transcript encoding a BCR light or heavy chain as described herein.Immune Profiling Methods in Single Cells or Single Nuclei

[0083] One aspect of the present disclosure relates to methods of labeling nucleic acids, e.g., labeling nucleic acids in a cell- or nucleus-specific manner. In some embodiments, the methods may comprise labeling nucleic acids in one or more B cells or B cell nuclei. Labeling or tagging methods according to the present disclosure may comprise, e.g.,: (a) generating complementary DNAs (cDNAs, or cDNA molecules) within a plurality of fixed and permeabilized B cells (or B cell nuclei) distributed into a plurality of aliquots or wells by reverse transcribing RNA molecules within the B cells (or nuclei) within the aliquots or wells, wherein the RNA molecules are reverse transcribed using reverse transcription (RT) primers, wherein at least some of the RT primers comprise a 5' overhang sequence, and wherein at least some of the RT primers comprise a well-specific barcode sequence; (c) pooling the plurality of B cells (or nuclei) dividing the plurality of B cells (or nuclei) into a number (n) of aliquots; (c) providing a plurality of nucleic acid tags to each of the n aliquots, wherein each labeling sequence of the plurality of nucleic acid tags provided into a given aliquot is the same, andwherein a different labeling sequence is provided into each of the n aliquots; (d) binding at least one of the cDNAs in each of the n aliquots to the nucleic acid tags; (e) combining the n aliquots; and (f) repeating steps (b), (c), (d), and (e) with the combined aliquot. In various embodiments, the plurality of B cells may be selected from eukaryotic cells, vertebrate cells, mammalian cells, mouse cells, and human cells. It will be appreciated that, as used herein, when a method is said to, e.g., label or tag nucleic acids “in” or “within” a cell or nucleus, this means that at least one of the labeling steps takes place at the interior of the cell or nucleus, but does not necessarily mean that all labeling or tagging steps take place at the interior of the cell or nucleus. For example, in some embodiments of the present methods, one or more tagging steps, such as those involving, e.g., reverse transcription and / ligation of a nucleic acid tag, may take place within cells or nuclei, and one or more subsequent steps (e.g., one or more tagging steps, e.g., as performed by ligation, polymerase extension, and / or amplification) may be performed following lysis of the cells or nuclei.

[0084] In certain embodiments, each nucleic acid tag may comprise a first strand including a 3' hybridization sequence extending from a 3' end of a labeling sequence and a 5' hybridization sequence extending from a 5' end of the labeling sequence. Each nucleic acid tag may also comprise a second strand including an overhang sequence. The overhang sequence may include (i) a first portion complementary to a 5' hybridization sequence (i.e., a 5’ hybridization sequence from a previously coupled nucleic acid tag) and / or to a 5' overhang sequence of a RT primer, and (ii) a second portion complementary to the 3' hybridization sequence. In some embodiments, the nucleic acid tag (e.g., the final nucleic acid tag, i.e., the final nucleic acid tag to be coupled to a cDNA molecule) may comprise a capture agent such as, but not limited to, a 5' biotin. A cDNA labeled with a 5' biotin-comprising nucleic acid tag may allow or permit the attachment or coupling of the cDNA to a streptavidin-coated magnetic bead. In some other embodiments, a plurality of beads may be coated with a capture strand (i.e., a nucleic acid sequence) that is configured to hybridize to a final sequence overhang of a barcode. In yet some other embodiments, cDNA may be purified or isolated by use of a commercially available kit (e.g., an RNEASY™ kit).

[0085] In various embodiments, step (f) (i.e., steps (b), (c), (d), and (e)) may be repeated a number of times sufficient to generate a unique combination or series of labeling sequences for the cDNAs in each B cell, i.e., that all the cDNAs originating from a given B cell will have the same combination or series of labeling sequences (also referred to as barcode sequences or index sequences), and that the complexity of the combinations or series of labeling sequencesis such that each combination or series is unique, or essentially unique, among all of the cells in the plurality of cells. For example, step (f) could be repeated enough times to generate a sufficient number of distinct combinations or series of labeling sequences that each individual combination or series has, e.g., at least a 95%, 96%, 97%, 98%, 99%, or higher probability of being unique among all of the combinations or series in the cells of the plurality. Stated another way, step (f) may be repeated a number of times such that the cDNAs in the first cell may have a first unique series of labeling sequences, the cDNAs in a second cell may have a second unique series of labeling sequences, the cDNAs in a third cell may have a third unique series of labeling sequences, and so on. The methods of the present disclosure may provide for the labeling of cDNA sequences from single cells with unique barcodes, wherein the unique barcodes may identify or aid in identifying the cell from which the cDNA originated. In other words, a portion, a majority, or substantially all of the cDNA from a single cell may have the same barcode, and that barcode may not be repeated in cDNA originating from one or more other cells in a sample (e.g., from a second cell, a third cell, a fourth cell, etc.).

[0086] In some embodiments, the present methods comprise (a) providing a plurality of permeabilized, fixed cells, wherein each of the plurality of permeabilized, fixed cells comprises RNA; (b) reverse transcribing RNA molecules within the cells to generate complementary DNA (cDNA) molecules, wherein the RNA molecules are reverse transcribed using reverse transcription (RT) primers each comprising: i) a poly(T) sequence or a random nucleotide sequence; and ii) a 5’ overhang sequence located 5’ of the poly(T) or the random nucleotide sequence, wherein the 5’ overhang sequence is the same in all of the RT primers used in the plurality of aliquots, and wherein following reverse transcription of the RNA the 5’ overhang sequence is present at the 5’ end of each of the cDNA molecules; (c) dividing the plurality of cells comprising the cDNA molecules into a plurality of aliquots, wherein each aliquot of the plurality of aliquots comprises more than one cell; (d) coupling nucleic acid tags to the cDNA molecules within cells of the plurality of aliquots, thereby generating tagged cDNA molecules, wherein each of the nucleic acid tags comprises: i) a barcode sequence, and ii) a 3’ hybridization sequence located 3’ of the barcode sequence and / or a 5’ hybridization sequence located 5’ of the barcode sequence, wherein multiple distinct barcode sequences are present among the nucleic acid tags used in the plurality of aliquots, and wherein the barcode sequences present among the nucleic tags used in each individual aliquot of the plurality of aliquots are specific to the individual aliquot; (e) combining the cells from the plurality of aliquots; (f) dividing the combined cells from the plurality of aliquots into a plurality of samples, whereineach sample of the plurality of samples comprises more than one cell; (g) lysing the cells in the plurality of samples to release the tagged cDNA molecules; and (h) amplifying the released tagged cDNA molecules in each of the plurality of samples using amplification primers, wherein at least a portion of the amplification primers used in each sample comprise an index sequence, wherein multiple distinct index sequences are present among the amplification primers used in the plurality of samples, and wherein the index sequences present among the amplification primers used in each individual sample of the plurality of samples are specific to the individual sample. In some embodiments, the method comprises preparing sequencing libraries from the isolated tagged cDNA molecules, e.g., a sequencing library comprising the whole transcriptome and a sequencing library directed specifically to BCR sequences (e.g., BCR heavy and / or light chains, including BCR heavy and light chain variable regions). In some embodiments, the BCR sequencing library is prepared by using hybrid capture to enrich BCR- specific sequences from among the amplified cDNA molecules.

[0087] In some embodiments, RT primers are used that include a BCR specific sequence. In some embodiments, steps (c) through (e) are repeated one or more times, thereby generating repeatedly tagged cDNA molecules. In some embodiments, steps (c) through (e) are repeated a number of times sufficient to generate at least as many distinct combinations of barcode sequences within the repeatedly tagged cDNA molecules as the number of cells in the plurality of cells. In some embodiments, steps (c) through (e) are repeated a number of times sufficient to provide a greater than 50%, 90%, 95%, or 99% probability that the combination of barcode sequences present among the repeatedly tagged cDNAs within a given individual cell is unique relative to the combinations of barcode sequences present among the repeatedly tagged cDNA molecules within the other cells of the plurality of cells.

[0088] In some embodiments, the coupling in step (d) comprises ligating a 3’ end of a nucleic acid tag to the 5’ end of a cDNA molecule. In some embodiments, the 3’ end of the nucleic acid tag is present within the 3’ hybridization sequence, and wherein the 3’ end of the nucleic acid tag is brought into proximity of the 5’ end of the cDNA molecule by being prehybridized to a linker nucleic acid strand that is complementary to the 3’ hybridization sequence of the nucleic acid tag and to the 5’ overhang sequence. In some embodiments, the coupling is stopped by introducing a plurality of ligation stop oligos that are complementary to all or part of the linker nucleic acid strand. In some embodiments, the coupling in step (d) during the one or more times that steps (c) to (e) are repeated comprises ligating a 3’ end of a nucleic acid tag to the 5’ end of a tagged or repeatedly tagged cDNA molecule. In someembodiments, the 3’ end of the nucleic acid tag is present within the 3’ hybridization sequence, and the 3’ end of the nucleic acid tag is brought into proximity of the 5’ end of the tagged or repeatedly tagged cDNA molecule by being prehybridized to a secondary linker sequence that is complementary to the 3’ hybridization sequence of the nucleic acid tag and to the 5’ hybridization sequence of a previously coupled nucleic acid tag. In some embodiments, the coupling is stopped by introducing a plurality of secondary ligation stop oligos that are complementary to all or part of the secondary linker sequence. In some embodiments, the RT primers and / or the nucleic acid tags are DNA molecules.

[0089] In some embodiments, the method further comprises prior to step (a), fixing and / or permeabilizing the plurality of cells. In some such embodiments, the fixation and / or permeabilization is performed at a temperature of, e.g., at or below about 8 °C, at or below about 7 °C, at or below about 6 °C, at or below about 5 °C, at or below about 4 °C, at or below about 3 °C, at or below about 2 °C, at or below about 1 °C, at or below about 0 °C, or on ice. Exemplary protocols for performing fixation and permeabilization of a wide variety of cell or nuclei quantities and throughputs are presented, e.g., in Examples 2 and 3.

[0090] In some embodiments, the nucleic acid tags that are coupled to the tagged or repeatedly tagged cDNA molecules during the last of the one or more times that steps (c) to (e) are repeated comprise a random nucleotide sequence to prevent counting of PCR duplicates, a capture agent, and / or a next-generation sequencing (NGS) adapter sequence. In some such embodiments, the capture agent comprises biotin. In some embodiments, the method further comprises, prior to step (h), isolating the tagged cDNA released during step (g) using streptavidin beads.

[0091] In some embodiments, the barcode sequences present within the nucleic acid tags each comprise at least 8 nucleotides. In some embodiments, one or more of the amplification primers further comprises one or more elements selected from the group consisting of a flowcell binding sequence, an NGS primer binding sequence, and an NGS adapter sequence.

[0092] In some embodiments, the cells are lysed in step (g) in the presence of a protease. In some embodiments, the protease is proteinase K. In some embodiments, following lysis, a protease inhibitor is added to the lysate and the tagged cDNA molecules are isolated from the lysate by binding them to a binding agent, e.g., streptavidin beads.

[0093] In some embodiments, the method further comprises: (i) sequencing the cDNA molecules amplified in step (h). In some embodiments, the method further comprises, prior to step (i), purifying the tagged cDNA molecules amplified in step (h) using solid-phase reversible immobilization (SPRI) beads. In some such embodiments, the tagged cDNA molecules are isolated from the lysate by binding them to a binding agent (such as streptavidin beads) in the presence of a protease inhibitor prior to purifying (i.e., size-selecting) the cDNAs using SPRI beads.

[0094] In some embodiments, the method further comprises (j) grouping the sequencing reads obtained in (i) by barcode sequence and / or index sequence. In some embodiments, the sequencing reads are grouped by a combination of barcode sequence and index sequence. In some embodiments, steps (c) to (e) have been repeated one or more times, thereby producing repeatedly tagged cDNA molecules each comprising multiple barcode sequences, and wherein the sequencing reads are grouped by any combination of two or more sequences from among the multiple barcode sequences and the index sequence.

[0095] It will be appreciated that, as used herein, when a method is said to, e.g., label or tag nucleic acids “in” or “within” a cell or nucleus, this means that at least one of the labeling steps takes place at the interior of the cell or nucleus (e.g., at least a first step or a first set of steps), but does not necessarily mean that all labeling or tagging steps take place at the interior of the cell or nucleus. For example, in some embodiments of the present methods, one or more tagging steps, such as those involving reverse transcription to generate cDNA molecules and the subsequent coupling of one or more nucleic acid tags to the cDNA molecules, may take place at the interior of the cells or nuclei, and one or more subsequent steps, such as template switching and preamplification / amplification steps, may be performed on tagged cDNA molecules isolated from the cells or nuclei following their lysis.

[0096] In some embodiments, the plurality of cells are mammalian cells. In some embodiments, a portion of the RT primers are specific to one or more BCR sequences. In some embodiments, the BCR sequences comprise sequences specific to BCR IgD, IgM, IgA, IgG, and / or IgE subtypes. In some embodiments, the BCR sequences comprise sequences specific to one or more BCR heavy chains, e.g., a BCR IgG, IgA, IgM, IgD, and / or IgE heavy chain. In some embodiments, the BCR sequences comprise sequences specific to one or more BCR light chains, e.g., a BCR kappa or lambda light chain. In some embodiments, the BCR sequences comprise sequences from a constant region of a BCR heavy or light chain. In someembodiments, the BCR sequences comprise sequences from a variable region of a BCR heavy or light chain. In some embodiments, the

[0097] In some embodiments, 96 distinct barcode sequences are present among the nucleic acid tags used in the plurality of aliquots. In some embodiments, the plurality of aliquots comprises 96 aliquots distributed in a 96-well plate. In some embodiments, each of the 96 distinct barcode sequences are present in only one of the 96 aliquots.

[0098] The barcodes used in the present methods, e.g., as present in RT primers or nucleic acid tags, can be any length, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the barcodes are at least 8 nucleotides long. In some embodiments, the barcodes are 8 nucleotides long. The length of any given barcode used in a given embodiment of the method (e.g., a barcode sequence in a first nucleic acid tag) can be independent of the length of a different barcode used in the embodiment (e.g., a barcode in a second nucleic acid tag). For example, in some embodiments a RT primer barcode, a barcode in a first nucleic acid tag, a barcode in a second nucleic acid tag, and / or a barcode or index sequence in a PCR primer could all be the same length, in some embodiments they could all be different lengths, in some embodiments some barcodes could be the same length and some could be different lengths.

[0099] In some embodiments, barcoded (or tagged) cDNA molecules can be mixed together and sequenced (e.g., using NGS), such that data can be gathered regarding RNA expression at the level of a single cell. For example, certain embodiments of the methods of the present disclosure may be useful in assessing, analyzing, or studying the transcriptome (i.e., the different RNA species transcribed from the genome of a given cell) of one or more individual cells. In particular embodiments, the transcriptome is studied together with the expression of BCR isoforms or clones in a plurality of individual single cells.

[0100] In particular embodiments, the cDNA molecules in the plurality of B cells (or nuclei derived from the B cells) include cDNA molecules derived from B-cell (BCR)-encoding mRNA in the cells. For example, in some embodiments, reverse transcription is performed within the B cells using primers designed to globally target RNA or mRNA within the B cells (i.e., target the transcriptome, or whole transcriptome, of the cells), such as primers including a poly(T) sequence (e.g., comprising 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive thymine bases) or comprising a random sequence (e.g., comprising 5, 6, 7, 8, 9, 10 or more random bases), as well as primers that specifically target BCR gene sequences (includingsequences from any BCR genes or encoding any one or more elements of a BCR, including, but not limited to, transcripts encoding any one or more of IgM, IgG, IgD, IgA, and IgE isotypes, IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2 subclasses, BCR heavy chain, BCR light chain, BCR heavy chain constant region, BCR heavy chain variable region, BCR light chain constant region, BCR light chain variable region, BCR mu heavy chain, BCR delta heavy chain, BCR gamma heavy chain, BCR epsilon heavy chain, BCR alpha heavy chain, BCR kappa light chain, BCR lambda light chain, BCR complementarity-determining region (CDR), BCR CDR1, BCR CDR2, BCR CDR3, BCR CDR-H1, BCR CDR-H2, or BCR CDR-H3 domain. As such, the cDNA molecules generated within a given B cell will include both enriched BCR gene transcripts and (non-enriched) non-BCR transcripts. In some embodiments, the RT primer targets a sequence at or close to the 3’ end of the BCR-encoding transcript, so as to obtain a full-length, or nearly full length transcript. In some embodiments, the RT primer targets an internal sequence within the BCR-encoding transcript.

[0101] As discussed above, for any one or more steps of the present methods, including, but not limited to, reverse transcription steps, nucleic acid tag coupling steps, cell lysis steps, template switching steps, amplification steps, or enrichment steps, an aliquot or group of cells (or nuclei, or lysates) can be separated into different reaction vessels or containers. Vessels or containers can also be referred to herein as receptacles, samples, and wells, and the terms vessel, container, receptacle, sample, and well may be used interchangeably herein. In some embodiments, cells or nuclei may be separated into a number of different reaction vessels. For example, the number of reaction vessels may include four 1.5 ml microcentrifuge tubes, a plurality of wells of a 96-well plate, or another suitable number and type of reaction vessels. In particular embodiments, the reaction vessels or containers include one or more 96-well plates (or, e.g., 6, 12, 24, 48, 384 well plates).

[0102] For combinatorial barcoding (or split-pool tagging), cells or nuclei can be distributed into a plurality of aliquots and polynucleotides within the cells or nuclei labeled with an aliquot-specific barcode (e.g., by reverse transcription of RNA within the cells or nuclei using primers comprising the barcode, or by appending a nucleic acid tag to polynucleotides within the cells or nuclei wherein the tags comprise aliquot-specific barcodes), the aliquots can then be repooled, washed, and separated again into a new plurality of aliquots, and a further set of barcodes can be added to the polynucleotides. In this way, after repeated rounds of separating, tagging, and repooling, cDNAs or other polynucleotides within each cell or nucleus may be bound to a unique combination or sequence of barcodes, or substantially uniquecombination or sequence of barcodes. In particular embodiments, all (or most, depending, e.g., on the efficiency of the tagging reactions in a given cell or nucleus) of the cDNA molecules or other polynucleotides within any individual cell or nucleus within a plurality of cells or nuclei will comprise the same combination of barcodes (or barcode sequences). In particular embodiments, the combination or sequence of barcodes can be used to identify, or help identify, the individual cell from which a given tagged cDNA molecule originated.

[0103] In particular embodiments, in a given barcoding step cells or nuclei within each well or aliquot are tagged with a different barcode, i.e., all of the barcodes (or barcode sequences) used within the well or aliquot are the same, while the barcode sequences are different in each of the wells or aliquots. However, other barcoding strategies are possible as well, e.g., in which more than one barcode sequence is used within a given well or aliquot, or in which one or more barcode sequences are present in multiple wells or aliquots during the barcoding step. In general, any barcoding protocol can be encompassed by the present disclosure so long that during the protocol the labeled molecules (e.g., RNA molecules, or cDNA molecules produced therefrom) within each cell or nucleus acquire a combination of barcodes that reflects the different wells or aliquots in which the cell or nucleus was present.

[0104] The different labeling sequences can be introduced at one or more steps, including during reverse transcription (e.g., wherein each reverse transcription (RT) primer comprises a barcode), during one or more subsequent labeling steps (e.g., ligating, tagmentation, or otherwise coupling a nucleic acid tag comprising a barcode sequence to a cDNA), or during one or more amplification steps (e.g., using one or more primers that include a barcode or index sequence). For example, in particular embodiments of the present disclosure, RNA is labeled within cells or nuclei by generating cDNA through reverse transcription (RT) using wellspecific barcode-containing primers, and subsequently additional well-specific barcodes are ligated to the cDNA molecules in one or more round of split-pool tagging, and finally yet more barcodes (or indexes) are added to the cDNA molecules during amplification using well- or sample-specific barcoded primers (e.g., unique dual indexes or UDIs). Accordingly, the number of possible barcode combinations can vary by, e.g., increasing or decreasing the number of wells used for reverse transcription, for ligation-based tagging, and / or for indexing during amplification, and / or by changing the number of total barcoding steps, e.g., by varying the number of rounds of split-pool tagging or by omitting barcodes in one or more steps (e.g., by performing RT and / or amplification using non-barcoded primers, or by omitting the ligation tagging steps and / or amplification indexing steps altogether).

[0105] In certain embodiments, steps of the present methods in which a nucleic acid tag is appended or coupled to a cDNA or other polynucleotide within a cell or nucleus may be repeated one or more times, e.g., 1, 2, 3, 4, 5 times, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 100, or more times. In certain other embodiments, the steps are repeated a sufficient number of times such that the cDNAs of each cell or nucleus would be likely to be bound to a unique barcode (e.g., unique among the plurality of cells or nuclei, or among multiple pluralities of cells or nuclei, e.g., in situations where multiple pluralities may be sequenced together). The number of times may be selected to provide a greater than 50% likelihood, greater than 90% likelihood, greater than 95% likelihood, greater than 99% likelihood, or some other probability that the cDNAs in each cell are bound to a unique barcode.

[0106] The number of total possible barcode combinations in the population will be a function of the number of barcode tagging rounds that are performed, and on the number of different barcodes / aliquots included in each round. The total number of possible barcode combinations can be achieved in any of a number of ways. In particular embodiments, the number of total possible barcode combinations is greater than the number of different cells in the population, e.g., such that the probability that a given combination of barcodes is unique among all of the cells of the plurality is, e.g., 95%, 96%, 97%, 98%, 99%, or higher. Accordingly, the present methods could include, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more individual rounds of barcoding.Cells and Nuclei

[0107] In some embodiments (e.g., embodiments related to immune profiling such as BCR profiling), the present methods can be used to specifically label molecules, e.g., nucleic acids, in any of a wide variety of immune cell types, particularly B cells (i.e., immune cells comprising a B-cell receptor (BCR)), or nuclei isolated from any of the herein-described cells. In some embodiments, the cells comprise non-B cells that express a BCR, e.g., B cell precursors, other immune cells, or any other cells expressing, or capable of expressing, a BCR (e.g., transgenic cells comprising a BCR-encoding expression construct). In some embodiments, the B cells (or other BCR-encoding cells) are mammalian cells. In some embodiments, the B cells (or other BCR-encoding cells) are human B cells. In some embodiments, the B cells (or other BCR-encoding cells) are mouse cells. In some embodiments, the B cells comprise chimeric BCRs, e.g., mouse B cells expressing BCRs with mouse constant regions and human variable regions. In some embodiments, the B cellscomprise activated B cells. In some embodiments, the B cells comprise primary B cells. In some embodiments, the B cells (or other BCR-encoding cells) comprise cells derived from a cell line. In some embodiments, the B cells (or other BCR-encoding cells) comprise cells derived from a tissue. In some embodiments, the B cells (or other BCR-encoding cells) comprise cells from a healthy individual. In some embodiments, the B cells (or other BCR- encoding cells) comprise cells from a subject with a disease.

[0108] In some embodiments, the B cells comprise immature B cells, mature B cells, transitional B cells, naive B cells, plasma B cells, and / or memory B cells. In some embodiments, the B cells comprise transitional B cells, naive B cells, memory B cells, plasma B cells, B cell progenitors, plasmablasts, plasma cells, lymphoplasmacytoid cells, follicular B cells, marginal-zone B cells, Bl cells, peripheral B cells, and / or regulatory B cells. The B cells can be obtained from any of a number of sources. In some embodiments, the B cells are isolated from a subject, e.g., a mammalian subject such as a human subject. In some embodiments, the B cells (or other BCR-encoding cells) are obtained from the bone marrow, spleen, lymph nodes, blood, lymph, secondary lymphoid organs, appendix, intestine, Peyer’s patches, tonsils, thymus, and / or lymphoid follicles of the subject.

[0109] In some embodiments, the cells are all from a single source, i.e., from a single individual, organism, or tissue. In some embodiments, the cells are from multiple sources, i.e. from multiple individuals, organisms, or tissues. In some embodiments, the cells are autologous cells. In some embodiments, the cells are allogeneic cells. In some embodiments, the cells are all or primarily comprise a single cell type (e.g., from a cell line, or a specific cell type isolated from a primary sample). In some embodiments, the cells comprise a mixture of different cell types. In some embodiments, the cells are adherent cells. In some embodiments, the cells are suspension cells.

[0110] In some embodiments, the cells have been previously frozen. In some embodiments, the cells have been previously fixed and frozen, e.g., the methods are performed using multiple samples that have been fixed and / or frozen at different times. In some embodiments, the cells have been previously fixed, permeabilized, and frozen.[OHl] In all of the herein-disclosed embodiments, either cells or nuclei obtained from the cells can be used. Nuclei can be prepared using known methods, e.g., by douncing. In some embodiments, nuclei are prepared from frozen cells, tissue samples, or tissue slices or sections. Nuclei can be prepared, e.g., by placing the frozen sample (cells, tissue, minced tissue sample,slice, section, etc.) into a cooled nuclei isolation (NIM) buffer solution (e.g., NIM1 or NIM2 buffer), then transferred to a dounce and homogenized, e.g., using a pestle (e.g., 10 strokes each with a loose and with a tight pestle). The homogenate can then be filtered (e.g., using a 40 um or 70 um filter) and transferred to, e.g. conical tubes. The tube can then be centrifuged, e.g., 200x or 500x g in a pre-cooled swinging bucket centrifuge for, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more minutes at a low temperature, e.g., 4 °C, or at 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, or 8 °C, or at a temperature less than 8 °C, 7 °C, 6 °C, 5 °C, 4 °C, 3 °C, 2 °C, or 1 °C. In some embodiments, the cells are counted before and / or after centrifugation, e.g., to ensure an appropriate number of cells in each aliquot or tube. The pellets can then be resuspended in an appropriate solution or buffer, e.g., a nuclei buffer containing BSA (e.g., 0.75% BSA), and subsequently fixed and stored, e.g., at -80 °C.

[0112] In particular embodiments of the present methods, at least 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,500,000, 2,000,000, 3,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, or more B cells or nuclei are used.

[0113] It will be appreciated that some embodiments of the present disclosure (e.g., embodiments related to low input cell or nuclei fixation), any of a large variety of cell types (or nuclei derived therefrom) can be used, including any of the cell types disclosed above (e.g., B cells or other BCR-expressing cells as disclosed herein), or any mammalian, human, mouse, animal, vertebrate, invertebrate, insect, fungal, plant, protist, plant, fungal, yeast, eukaryotic, prokaryotic, bacterial, or other cells or nuclei. For example, the herein-disclosed low-input fixation methods can be used along with protocols to specifically label molecules, e.g., nucleic acids, in any of a wide variety of cell types (or nuclei derived therefrom), including primary cells, cell lines (e.g., HEK293, HEK293T, HEK293F, NIH3T3, Jurkat cells, or others), cells isolated from an organism, organoid, or a tissue, isolated blood cells, healthy cells (e.g., wildtype or control cells), diseased cells (e.g., cancer cells, infected cells), stem cells, pluripotent cells,. It will be appreciated that any of the present methods can be equally used for either cells or nuclei. Accordingly, at any location of the present disclosure in which cells are referred to, it can be considered that the disclosure applies equally to nuclei, even if nuclei are not explicitly mentioned.

[0114] In some embodiments, the cells (or nuclei) used in the herein-disclosed methods comprise one or more cell types such as blood cells (e.g., peripheral blood mononuclear cellsor PBMCs, immune cells such as T cells, B cells, NK cells), brain cells, liver cells, gut cells, bone marrow cells, pancreatic cells, epithelial cells, endothelial cells, neuronal cells, fibroblast cells, bone cells, muscle cells, skin cells, fat cells, lymphocytes, myeloid cells, macrophages, stem cells, and others. In some embodiments, the cells are all from a single source, i.e., from a single individual, organism, or tissue. In some embodiments, the cells are from multiple sources, i.e. from multiple individuals, organisms, or tissues. In some embodiments, the cells are autologous cells. In some embodiments, the cells are allogeneic cells. In some embodiments, the cells are all or primarily comprise a single cell type (e.g., from a cell line, or a specific cell type isolated from a primary sample). In some embodiments, the cells comprise a mixture of different cell types. In some embodiments, the cells are adherent cells. In some embodiments, the cells are suspension cells.Fixation and permeabilization

[0115] In some embodiments, the present methods comprise fixing and permeabilizing the plurality of B cells or nuclei prior to, e.g., generating cDNA in the cells or nuclei by reverse transcribing RNA in the cells or nuclei. In some embodiments, the B cells may be fixed and permeabilized and frozen, e.g., at -80 °C, prior to, e.g., generating cDNA. In some embodiments, the B cells are fixed and permeabilized and then directly used in the present methods, i.e., without freezing and storing them.

[0116] The plurality of cells (or nuclei) may be fixed using any of a number of suitable reagents or conditions. For example, in some embodiments, the cells (or nuclei) can be fixed in formaldehyde in phosphate buffered saline (PBS) (e.g., in about 1-4% formaldehyde in PBS). In various embodiments, the plurality of cells (or nuclei) may be fixed using methanol (e.g., 100% methanol) at about -20° C. or at about 25° C. In various other embodiments, the plurality of cells (or nuclei) may be fixed using methanol (e.g., 100% methanol), at between about -20° C. and about 25° C. In yet various other embodiments, the plurality of cells (or nuclei) may be fixed using ethanol (e.g., about 70- 100% ethanol) at about -20° C. or at room temperature. In yet various other embodiments, the plurality of cells (or nuclei) may be fixed using ethanol (e.g., about 70-100% ethanol) at between about -20° C. and room temperature. In still various other embodiments, the plurality of cells (or nuclei) may be fixed using acetic acid, for example, at about -20° C. In still various other embodiments, the plurality of cells (or nuclei) may be fixed using acetone, for example, at about -20° C. Other suitable methods of fixing the plurality ofcells (or nuclei) are also within the scope of this disclosure. Other suitable fixatives that can be used alone or in combination include phosphate buffered formalin, formal calcium, formal saline, zinc formalin (unbuffered), Zenker’s fixative, Helly’s fixative, B-5 fixative, Bouin’s solution, Hollande’s, Gendre’s solution, Clarke’s solution, Carnoy’s solution, methacam, alcoholic formalin, formol acetic alcohol, formaldehyde, paraformaldehyde, glutaraldehyde, formalin, acrolein, glyoxal, osmium tetroxide, carbodiimides, diimidoesters, chloro-s-triazides, diisocyanates, diethylpyrocarbonate, maleimides, benzoquinone, mercuric chloride, zinc chloride, zinc sulphate, picric acid, potassium dichromate, ethanol, methanol, acetone, acetic acid, and others.

[0117] In some embodiments, RNases are inactivated or eliminated before, during, and / or after fixation and / or permeabilization using, e.g., RNase decontamination products such as RNaseZap RNAse Decontamination Solution (Thermo Fisher). In some embodiments, BSA (e.g., 5-10%, or 7.5%) is added to the cells or nuclei, e.g., to prevent aggregation.

[0118] In some embodiments, the methods may include fixing and / or permeabilizing the cells or nuclei at a temperature below about 8 °C, below about 7 °C, below about 6 °C, below about 5 °C, below about 4 °C, below about 3 °C, below about 2 °C, below about 1 °C, below about 0 °C, below about -5 °C, below about -10 °C, at about 8 °C, at about 7 °C, at about 6 °C, at about 5 °C, at about 4 °C, at about 3 °C, at about 2 °C, at about 1 °C, or at another suitable temperature. In particular embodiments, the B cells are fixed and / or permeabilized at a temperature of below about 8, 7, 6, 5, 4, 3, 2, 1, 0, -1, -2, -3, or -4 °C, between about -4 to 8, - 4 to 0, 0 to 4, 4 to 8, or 0 to 8 °C, or at about 8, 7, 6, 5, 4, 3, 2, 1, 0, -1, -2, -3, or -4 °C.

[0119] In some embodiments, the cells are adherent cells (i.e., cells that are adhered to a plate, e.g., adherent mammalian cells). In some such embodiments, adherent cells are fixed, permeabilized, and / or undergo reverse transcription, followed by trypsinization to detach the cells from a surface. Alternatively, the adherent cells may be detached prior to the separation and / or tagging steps. In some other embodiments, the adherent cells may be trypsinized prior to the fixing and / or permeabilizing steps.

[0120] Permeabilization of the cells (or nuclei) can be achieved in any of a number of ways. For example, a detergent or surfactant such as TRITON™ X-100 may be added to the plurality of cells (or nuclei), followed by the optional addition of HC1. In some such embodiments, about 0.2% TRITON™ X-100 is added to the plurality of cells (or nuclei), followed by the addition of about 0.1 N HC1. In some embodiments, the plurality of cells (ornuclei) is permeabilized using ethanol (e.g., about 70% ethanol), methanol (e.g., about 100% methanol), Tween 20 (e.g., about 0.2% Tween 20), and / or NP -40 (e.g., about 0.1% NP-40).

[0121] In some embodiments, the cells (or nuclei) are fixed by following, or substantially following, the exemplary protocol described in Example 2 and shown in FIG. 14 or FIG. 16, i.e., involving a workflow involving: i) centrifuging cells (or nuclei) (e.g., 100,000-1,000,000 cells or nuclei) and removing the supernatant); ii) resuspending the cells (or nuclei) in a prefixation mix and straining the cells (or nuclei) into a new tube; iii) adding a fixative master mix to the cells (or nuclei) and incubating on ice for, e.g., 10 minutes; adding a permeabilization solution and incubating on ice for, e.g., 3 minutes,; iv) adding a fix and permeabilization stop buffer; v) centrifuging the fixed and permeabilized cells (or nuclei) at, e.g., 200-500 x g at, e.g., 4 °C and then removing the supernatant; vi) resuspending the cells (or nuclei) in a cell storage master mix and straining the cells (or nuclei) into a new tube; and vii) counting the fixed cells and optionally aliquoting them into samples before freezing at -80 °C (or proceeding directly into barcoding steps).Low-input fixation

[0122] In one aspect of the present disclosure, cells or nuclei are fixed and permeabilized using a modified protocol that involves the use of cell (or nuclei) binding magnetic beads such as concanavalin A (or ConA) beads. Such protocols can be used for any of the single-cell immune profiling methods described herein (e.g., for BCR profiling), as well as for any other type of immune profiling (e.g., TCR profiling) or any other scRNA-seq application (e.g., whole transcriptome analysis, CRISPR screens, gene capture) or sc gDNA-seq approaches such as ATAC-seq, DNase-seq, and others).

[0123] Such low-input methods can be used, e.g., to fix low numbers of cells (e.g., from 10,000-100,000 cells or nuclei in one reaction, divided in up to 12 samples (e.g., up to 12 tubes) or up to 96 samples (e.g., in a 96-well plate), such that relatively few cells or nuclei may be present in each tube or well and maintaining a high level of retention of the cells or nuclei in each tube or well is of high importance. It will be appreciated that such methods can be scaled up, e.g., when fixing and barcoding a large number of cells or nuclei divided into a large number of individual samples (e.g., up to 1,000,000 cells or nuclei divided across 384 different samples or experimental conditions), such that the number of cells or nuclei in each sample is still low and it is of high important to maintain the highest possible retention rate in each sample.

[0124] The present disclosure provides methods and kits involving the use of beads, e.g., magnetic beads comprising affinity to cells or nuclei such as ConA (concanavalin A) beads. Using ConA beads in protocols such as for single-cell sequencing, e.g., single-cell RNA sequencing or methods of single cell DNA sequencing such as scDNase-seq, scMNase-seq, or scATAC-seq. The present methods provide numerous advantages, in particular relating to cell / nuclei handling and retention, that allow, e.g., such methods to be performed with low numbers of cells or nuclei, while maintaining high rates of cell / nuclei retention and without impacting the quality or efficiency of labeling (e.g., RNA or gDNA labeling).

[0125] In some embodiments, the present disclosure includes workflows designed to efficiently process between, e.g. 10,000 and 100,000 cells or nuclei, accommodating up to 12 samples or as many as 96 samples simultaneously. The fixation protocol preserves cell (or nuclear) structure, prevents, e.g., RNA degradation, and locks desired molecules such as RNA and / or DNA fragments inside the cells, essential for downstream processing with Evercode's split-pool combinatorial barcoding technology.

[0126] The present low-input fixation methods can be used, e.g., to improve cell / nuclear handling and retention for any of a number of steps in split-pool barcoding methods. In some embodiments, the methods involve the use of beads in a fixation and / or permeabilization step. In some embodiments, the methods involve the use of beads in an enzymatic processing step such as reverse transcription or genomic DNA fragmentation. In some embodiments, the methods involve the use of beads in one or more steps of a split-pool barcoding protocol. In any of the herein disclosed embodiments, the beads can provide any of a number of advantages including, but not limited to, reduced (or eliminated) need for centrifugation, simplifying buffer exchange, and allowing lower resuspension volumes. Such advantages apply to any cell or nuclei sample, including samples with low cell numbers, in which low cell retention rates can be particularly problematic, and in which low resuspension volumes can be desirable, e.g., to maintain a relatively high final concentration of cells or nuclei (e.g., high enough for barcoding protocols such as split-pool barcoding). Such methods can be used, e.g., with rare samples (e.g., clinical or biological samples) or with experiments involving high numbers of experimental conditions (e.g., in a screen) such that only a small number of cells is present for each individual condition.

[0127] In some embodiments, protocols are used for cell or nuclei fixation. Using such protocols, fixed samples are stable for up to 4 months, or longer, at -80°C, providing flexibilityby decoupling sample collection from library preparation. This allows samples to be stored and batched post-fixation, enabling simultaneous library preparation and minimizing batch effects. The workflow facilitates parallel fixation of multiple samples, streamlining the process when handling up to 96 samples at a time.

[0128] The present methods can be used as part of, or in combination with, any of a variety of RNA or DNA labeling methods, e.g. split pool (combinatorial) barcoding methods for transcriptomic or genomic DNA analysis, such as methods described in, e.g., in US Patent Nos. 10,900,065, 11,634,751, 11,168,355, 11,427,856, 11,555,216, 11,639,519, 12,043,864, 11,987,838, 11,680,283, 10,633,648, 11,421,221, US Pat. App. Pub. No. US 2021 / 0388415 Al, 20230265487A1, 20230392192A1, 20230392193A1, 20230399680A1, 20240002907A1, 20240110227A1, 20240110226A1, US20230304073A1, US20230340567A1,US20240240232A1, US20240240233A1, 20230014658 Al, US20210388415A1,20220162691A1, 20240182964A1, 20240200132A1, in Rosenberg et al., Science 360, 176- 182 (2018), Rosenberg etal., BioRxiv (2017), “Scaling single cell transcriptomics through split pool barcoding,” doi.org / 10.1101 / 105163, and Tran et al. BioRxiv (2022) “High sensitivity single cell RNA sequencing with split pool barcoding,” doi.org / 10.1101 / 2022.08.27.505512, the entire disclosures of all of which are herein incorporated by reference (including all supplemental material).

[0129] In some embodiments, the low-input fixation protocol is performed according to, or substantially according to, the exemplary low-input fixation and permeabilization protocol described in Example 3 and shown in, e.g., FIG. 17, i.e., a protocol involving: i) centrifuging cells or nuclei present in 20 L of buffer or media (in tubes or multi-well plates), and removing the supernatant; ii) adding prefixation buffer and straining the cells or nuclei; iii) adding cell / nuclei fixation master mix, and incubating on ice (e.g., for 10 minutes); iv) adding permeabilization solution is added, and incubating on ice (e.g., for 3 minutes); v) adding a fixation and permeabilization stop solution; freezing the cells or nuclei at -80 °C; vi) thawing the tubes or plates; vii) adding cell-binding beads (e.g., ConA beads) to each tube or well, and mixing; viii) placing the tubes or plates on a magnet (e.g., for 5 minutes), and removing the supernatant; ix) resuspending the cells or nuclei in storage buffer; and x) proceeding with a single-cell combinatorial barcoding protocol such as Evercode WT, mini or mega, BCR, TCR, CRISPR, gene capture, DNase-seq, ATAC-seq, or others.

[0130] The low input fixation protocol can be performed on any of a range of quantities of cells or nuclei, e.g., 1-10,000; 10-100,000; 100,000-1,000,000; 1,000,000-10,000,000, 10,000,000-100,000,000, or 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, or more cells or nuclei, and / or the cells or nuclei can be divided into any number of different samples (e.g., corresponding to different cell types, experimental conditions, subjects, etc.), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 24, 32, 36, 40, 48, 56, 60, 64, 70, 72, 76, 80, 84, 88, 94, 96, 192, 384, or more samples.

[0131] In some embodiments, an amount of beads is added to each aliquot of cells or nuclei such that a high retention level (e.g., the number of cells or nuclei present at the end of the fixation and permeabilization protocol, or the number of cells or nuclei entering into a barcoding protocol, or the number of cells or nuclei present at any step within a barcoding protocol, or the number of cells or nuclei present at the end of a barcoding protocol, relative to the number of cells or nuclei inputted into the fixation protocol, i.e., the number present at the beginning of the protocol. In some embodiments, the retention rate is at least 50%, or at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some embodiments, the retention rate is higher for large cells or nuclei than for small cells or nuclei (e.g., in some embodiments the retention rate is at least 65% for small cells or nuclei and at least 80% for large cells or nuclei). Retention rates can be determined by, e.g., counting cells at any of a number of possible points in the protocol, including at various points described herein, and can be carried out using, e.g., a hemocytometer or other suitable cell counting device, or can be estimated based on guidance provided herein or based on observations or knowledge of one of ordinary skill in the art for, e.g., a given cell type or set of experimental conditions.

[0132] In some embodiments, an amount of beads is added to each aliquot of cells or nuclei such that a low multiplet rate is observed and / or minimal clumping of cells is observed. For example, in some embodiments, if clumping or multiplets are observed, an increased number of beads can be added which may alleviate the clumping or multiplets. In some embodiments, other optimizations are performed, such as optimizing centrifugation conditions as described elsewhere herein. Multiplet and / or clumping rates can be determined, e.g., by visual inspection of cells or nuclei under a microscope (see, e.g., FIGS 9A-9C) or through analysis of sequencing results, as described in more detail elsewhere herein.

[0133] In some embodiments, an amount of beads is added to each aliquot or nuclei such that a maximum amount of beads is not exceeded such that the beads may impact downstream enzymatic steps such as reverse transcription. Such optimization can be assessed, e.g., by analyzing sequencing reads following barcoding, and is within the skill of one or ordinary skill in the art.

[0134] In some embodiments, an amount of beads is added according to the ratios and / or at a volume shown in Table 63 or Table 88. In some embodiments, at least 4 mL of beads is added per 50,000 cells or nuclei, or at least 4 mL of beads for each 14,000 cells or nuclei, or at least 6 mL of beads for each 50,000 cells or nuclei, or at least about 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL or more beads is added for each 10,000 cells or nuclei, or about 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL or more beads is added for each 50,000 cells or nuclei, or at least about 4 mL, 5 mL, 6 mL, 7 mL, 8 mL 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, or more is added for each 100,000 cells or nuclei. In some embodiments, the amount of resuspension buffer added to the cells and beads is at least about 3 times the volume of beads added, or at least about 3, 3.5, 4, 4.5, 5 or more times the volume of beads added (see, e.g., Table 88). In some embodiments, an adequate volume of buffer is added along with the beads to the cells or nuclei, e.g., as indicated in Table 88, so as to avoid attained excessive concentrations of beads that could affect downstream steps such as reverse transcription.

[0135] In some embodiments, the beads are bound to cells or nuclei in one or more tubes or in the wells of a plate, e.g., a multi -well plate such as a 96-well or 384-well plate. In some embodiments, the plate is a deep-well plate. In some embodiments, the plate is a PCR plate. In some embodiments, the wells of the plate have a round bottom, a flat bottom, or a conical bottom. In some embodiments, the cells are polypropylene plates.

[0136] One or more magnets are used at various steps of the present methods, in particular to introduce a localized magnetic force to the sides of the tubes or wells containing the cells or nuclei and the beads. For example, magnets can be applied to the bottoms or sides of a well or tube so as to attract the bead-bound cells or nuclei in the well or tube and hold them in place to facilitate, e.g., supernatant removal or buffer exchange, e.g. in the absence of centrifugation. In some embodiments, magnetic separation racks are used, e.g., comprising one or more magnet, or magnet plates are used (e.g., 96-well or 384-well magnet plates) available from commercial suppliers, including in certain of Parse Bioscience’s Evercode fixation kits.

[0137] It will be appreciated that in all of the herein-disclosed embodiments, either cells or nuclei obtained from the cells can be used. Nuclei can be prepared, e.g., using standard methods such as by douncing. In some embodiments, nuclei are prepared from frozen cells, tissue samples, or tissue slices or sections. In some embodiments, nuclei are obtained from fresh cells, e.g., in embodiments in which the beads are used to fix, permeabilize, and freeze nuclei.

[0138] The present methods involve the use of magnetic beads that comprise affinity to the surface of cells or nuclei. In particular embodiments, the beads are coated with concanavalin A (ConA). ConA beads that are suitable for use in the present methods can be prepared for use in the methods or can be obtained from commercial suppliers such as VectorLabs, Cell Signaling Technology, EpiCypher, APExBIO, Sigma Aldrich, AMSBIO, and others.Reverse transcription

[0139] In particular embodiments, reverse transcription is conducted or performed on the plurality of B cells or nuclei. In certain embodiments, reverse transcription may be conducted on a fixed and / or permeabilized plurality of B cells (or nuclei). In some embodiments, variants of M-MuLV reverse transcriptase may be used in the reverse transcription. However, any suitable method of reverse transcription is within the scope of this disclosure. An exemplary overview of reverse transcription is shown, e.g., in FIG. 2A.

[0140] In some embodiments, a portion of a reverse transcription (RT) primer that is configured to bind to RNA and / or initiate reverse transcription may comprise one or more of the following: a random hexamer, random septamer, an octamer, a nonamer, a decamer, or a poly(T) (or polydT) stretch of nucleotides (e.g., comprising 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive thymine bases). In some embodiments, the poly(T) sequence is anchored, i.e., comprises a base other than T at the 3’ end (e.g., a mixture of anchored primers is used each comprising an A, C, or G at the 3’ end of the poly(T) sequence). In particular embodiments, RT primers are used that comprise well-specific barcodes, as described in more detail elsewhere herein.

[0141] In some embodiments, primers comprising a poly(T) sequence are used in the reverse transcription in the absence of primers with random sequences (such as random hexamers). In some embodiments, primers comprising random sequences (e.g., random hexamers) are used in the absence of primers with poly(T) sequences. In particularembodiments, the reverse transcription is performed with both primers comprising a random sequence (e.g., random hexamer) and primers comprising a poly(T) sequence (e.g., with 15 consecutive thymidine residues).

[0142] In particular embodiments, the RT primers also include specific primers configured to enrich for specific transcripts among the population of cells, e.g., transcripts corresponding to genes encoding B cell receptors (BCRs), corresponding to other immune related genes, or to other genes whose activity is potentially influenced by BCR activity (e.g., genes involved in signal transduction, transcriptional regulation, cell proliferation, or any other activity induced or inhibited by BCR activation).

[0143] In some embodiments, RT primers are used that correspond a gene encoding a BCR, e.g., to a constant region of a BCR-encoding gene. In some embodiments, RT primers are used that correspond to (i.e., are configured to reverse transcribe) one or more BCR chains, e.g., to one or more BCR IgM, IgG, IgD, IgA, and / or IgE isotypes or heavy chains, and / or to kappa and / or lambda light chains. In some embodiments, the RT primers used comprise at least one primer specific to an IgM heavy chain, at least one primer specific to an IgD heavy chain, at least one primer specific to an IgG heavy chain, at least one primer specific to an IgA heavy chain, and at least one primer specific to an IgE heavy chain. In some embodiments, the RT primers further comprise at least one primer specific to a Ig kappa light chain, and at least one primer specific to an Ig lambda light chain.

[0144] In some embodiments, each of the RT primers further comprises a 5’ overhang comprising a 5’ overhang sequence located 5’ of the poly(T) or the random nucleotide sequence, wherein the 5’ overhang sequence is the same in all of the RT primers used in the first plurality of aliquots, and wherein following reverse transcription of the RNA, the 5’ overhang sequence is present at the 5’ end of each of the cDNA molecules.

[0145] In some embodiments, the concentration of each RT primer (e.g., a polydT primer, a random hexamer primer, or other primer such as a BCR-specific or other gene-specific primer) is between about 0.5 pM and about 10 pM. In some embodiments, the concentrations are each between about 1 pM and about 7 pM, between about 1.5 pM and about 4 pM, between about 2 pM and about 3 pM, about 2.5 pM, or another suitable concentration.

[0146] In some embodiments, each of the RT primers comprises a barcode (i.e., a specific barcode sequence) (i.e., an “RT primer barcode”). In particular embodiments, the reversetranscription is performed on a population of B cells (or nuclei) distributed in a plurality of aliquots or wells, and the RT primer barcodes are aliquot- or well-specific. In some such embodiments, all of the RT primer barcodes used in a given aliquot or well are the same, and a different RT primer barcode is used in each of the aliquots or wells. Stated another way, a first barcode may be added to the cDNA molecules in a first specific container, mixture, reaction, receptacle, sample, well, or vessel (e.g., specific to the given container, mixture, reaction, receptacle, sample, well, or vessel), and a second barcode sequence may be added to the cDNA molecules in a second container, mixture, reaction, receptacle, sample, well, or vessel (and the same for a third, fourth, etc. barcode sequence and third, fourth, etc. container, mixture, reaction, receptacle, sample, well, or vessel). For example, in some embodiments, 48 sets of different well-specific RT primers are used (e.g., in a 48-well plate). Accordingly, if there are 48 samples (e.g., cells, tissues, etc.), each sample can get a unique well-specific barcode. However, if there are only four samples, each sample can have 12 different sets of well-specific RT primers. A user can know which 12 correspond to each sample, so the user can recover sample identities. Other numbers of specific barcodes (or well-specific RT primers) are also within the scope of this disclosure. Such a configuration may allow or provide for the multiplexing of the method. In general, any distribution of barcode sequences that can provide some information about the aliquot or well in which a given cell was present can be used. For example, even if more than one barcode sequence is used in one or more aliquots or wells, or if a given barcode sequence is used in more than one aliquot or well, the methods are encompassed by the present disclosure.

[0147] The barcode sequences present within the RT primers can have any of a range of lengths, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In particular embodiments, the RT primer barcode sequences are 8 nucleotides in length. By varying 8 nucleotides, there are 65,536 possible unique sequences. In some embodiments, the RT primer barcodes comprise more than 8 nucleotides. In some other embodiments, the RT primers comprise fewer than 8 nucleotides.

[0148] In some embodiments, the reverse transcription primers may be configured to reverse transcribe all, or substantially all, RNA in a cell (e.g., a random hexamer with a 5' overhang). In some other embodiments, the reverse transcription primers may be configured to reverse transcribe RNA having a poly(A) tail (e.g., a poly(dT) primer, such as a dT(15) primer, with a 5' overhang). In particular embodiments, the reverse transcription primers are configured to reverse transcribe both all, or substantially all, RNA in a cell, as well as to reverse transcribepolyadenylated RNA. In some embodiments, reverse transcription primers may be configured to reverse transcribe predetermined RNAs (e.g., a set of BCR-specific primers, as described above, or one or more genes downstream of or otherwise response to BCR activation, one or more genes involved in any aspect of an immune response, or any one or more genes of interest in relation to BCR activity and / or an immune response).

[0149] The plurality of cells can be divided prior to reverse transcription into any of a number of aliquots or wells, and using any of a number of suitable reaction vessels or containers. For example, the plurality of cells or nuclei can be distributed into individual tubes or containers, or into a plurality of wells in a multi-well plate. Any multi-well plate can be used, e.g., 4, 6, 8, 12, 24, 48, 96, 384, or 1536 well plates. In particular embodiments, the plurality of cells or nuclei are distributed into one or more 96-well plates. In some embodiments, all 96 wells of a 96-well plate are used (i.e., the plurality of cells or nuclei is divided into 96 aliquots). In some embodiments, a fraction of the wells on the plate are used, e.g., the cells or nuclei are distributed into, e.g., 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 12, 24, 36, 48, 60, 72, or 84 wells of a 96-well plate. In particular embodiments, up to 12 wells are used for up to 10,000 cells or nuclei, or up to 48 wells are used for up to 100,000 cells or nuclei, or up to 96 wells are used for up to 1,000,000 cells or nuclei.

[0150] Suitable methods of reverse transcribing RNA within cells for use in the herein- described methods are described, e.g., in US Patent Nos. 10,900,065, 11,634,751, 11,168,355, 11,427,856, 11,555,216, 11,639,519, 11,680,283, 10,633,648, 11,421,221, US Pat. App. Pub. No. US 2021 / 0388415 Al, in Rosenberg etal., Science 360, 176-182 (2018), Rosenberg etal., BioRxiv (2017), “Scaling single cell transcriptomics through split pool barcoding,” doi.org / 10.1101 / 105163, Tran et al. BioRxiv (2022) “High sensitivity single cell RNA sequencing with split pool barcoding,” doi.org / 10.1101 / 2022.08.27.505512, the entire disclosures of all of which are herein incorporated by reference (including all supplemental material).

[0151] In particular embodiments, one or more of the RT primers used comprise a nucleotide sequence as shown in any of Tables 1-6. Tables 1-6 provide primer sequences suitable, e.g., for the wells of a 96-well plate, with each primer sequence comprising a constant sequence, a barcode, and a primer sequence. Primers used in the present methods can comprise any of these elements, alone or in combination, e.g., the primer sequence with a different barcode and / or a different constant sequence.Multiomics applications

[0152] In some embodiments, the herein described immune profiling methods (e.g., examining the whole transcriptome as well as BCRs in parallel), are included in multiomics approaches in which other aspects of the cells or nuclei are also analyzed, e.g., in DNase-seq, whole genome sequencing, targeted genome sequencing, ChlP-sequencing, ATAC-seq, or other multi-omics methods to obtain information relative to the genotype, gene activity, protein activity, or other aspects of the cells in conjunction with the information obtained relative to immune profiling and transcriptome as described elsewhere herein.

[0153] In certain embodiments, other molecules such as proteins, peptides, and / or antigens, can be labeled combinatorially in conjunction with the labeling of the whole transcriptome and specific target (e.g., gRNA) as described herein. For example, the molecule can be bound by, e.g., an antibody or aptamer that specifically binds to the molecule, wherein the antibody or aptamer is conjugated with an adapter sequence (e.g., a nucleic acid adapter sequence comprising a barcode sequence such as a well-specific barcode sequence). The barcode sequence may be configured to uniquely identify the molecule to which the antibody or adapter is bound.

[0154] Methods related to binding or coupling an adapter sequence to a protein, a peptide, and / or an antigen may be used, for example, in protein quantification, peptide quantification, and / or antigen quantification. In various embodiments, the adapter sequence can be attached (e.g., chemically attached) to the antibody or aptamer (e.g., a peptide aptamer). For example,the adapter sequence can be attached to an antibody or aptamer using chemistry known to the skilled artisan for mediating DNA -protein bonds. Antibodies for different proteins can be labeled with nucleic acid sequences or strands that include a unique identifier sequence in addition to the adapter sequence. The antibody, or set of antibodies, may then be used in an immunostaining experiment to label a protein, or set of proteins, in fixed and / or permeabilized cells or tissue. Subsequently, the cells may undergo combinatorial (i.e., split-pool) labeling or barcoding as disclosed herein.

[0155] In some embodiments, the RNA can be labeled with adapter sequence using hybridization, for example, via Watson-Crick base-pairing. After the labeling steps and / or cell lysis, as discussed above, for example, the adapter sequence may be configured to prime reverse transcription to form or generate cDNA .Labeling with nucleic acid tags (Combinatorial bar coding)

[0156] In some embodiments, barcoded (or tagged) cDNA is mixed together and sequenced (e.g., using NGS), such that data can be gathered regarding RNA expression at the level of a single immune cell. For example, the methods of the present disclosure may be useful in assessing, analyzing, or studying the whole transcriptome (i.e., all of the different RNA species transcribed from the genome of a given cell, or all of the mRNA molecules expressed in a given cell) of one or more individual immune cells, together with the identification of the specific B cells receptors expressed in the cells (i.e., with the BCR clonotype of the individual cell). An exemplary overview of such tagging is shown, e.g., in FIGS. 2B-2C, and an illustration of nucleic tags that can be used in certain embodiments of the present methods is shown, e.g., in FIGS. 8A-8C.

[0157] As described above, in particular embodiments of the present disclosure a plurality of cells or nuclei is subjected to combinatorial barcoding, such that molecules (e.g., cDNAs synthesized in cells by reverse transcription) are labeled with barcodes that when viewed in combination provide a cell- or nucleus-specific label for the labeled molecules. In such labeling steps, a plurality of cells or nuclei are divided into multiple aliquots or wells, labeled with wellspecific barcodes (e.g., during reverse transcription using barcoded primers, by ligating barcoded nucleic acid tags to cDNA or tagged cDNA molecules), and repooled. This cycle of dividing the plurality of cells, well-specific labeling, and repooling can be repeated any number of times, with each round adding more tags to the cDNAs and thereby creating a set of nucleic acid tags that together can act as, e.g., a cell-specific (or nucleus-specific) barcode. As moreand more rounds are added, the number of paths that a cell can take increases, and consequently the number of possible barcodes that can be created also increases. Given enough rounds and divisions, the number of possible barcodes will be much higher than the number of cells, resulting in a high likelihood that each cell (or nucleus) in the population has a unique barcode. For example, if the division took place in a 96-well plate, after 4 divisions there would be 964=84,934,656 possible barcodes. In particular embodiments of the present methods, enough rounds of labeling is performed such that the number of possible barcodes is 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or lx, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx, or more of the number of cells (or nuclei) in the plurality of cells (or nuclei). In particular embodiments, enough rounds of labeling are performed such that the likelihood that the tagged molecules within a given cell or nucleus have a unique barcode combination among the plurality of cells is about 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or greater.

[0158] In each round of tagging, the plurality of cells or nuclei can be divided into any of a number of aliquots or wells, and using any of a number of suitable reaction vessels or containers. For example, the plurality of cells or nuclei can be distributed into individual tubes or containers, or into a plurality of wells in a multi-well plate. Any multi-well plate can be used, e.g., 4, 6, 8, 12, 24, 48, 96, 384, or 1536 well plates. In particular embodiments, the plurality of cells or nuclei are distributed into one or more 96-well plates. In some embodiments, all 96 wells of a 96-well plate are used (i.e., the plurality of cells or nuclei is divided into 96 aliquots). In some embodiments, a fraction of the wells on the plate are used, e.g., the cells or nuclei are distributed into, e.g., 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 12, 24, 36, 48, 60, 72, or 84 wells of a 96-well plate. In some embodiments, 96 distinct barcode sequences are present among the nucleic acid tags used in the plurality of aliquots. In some embodiments, the plurality of aliquots comprises 96 aliquots distributed in a 96-well plate. In some embodiments, each of the 96 distinct barcode sequences are present in only one of the 96 aliquots.

[0159] FIGS. 2A-2C illustrate an exemplary embodiment of labelling or tagging with nucleic tags. As shown, during reverse transcription of the mRNA molecules (FIG. 2A), Poly T and random hexamer primers anneal to mRNA within single cells. In some embodiments, primers specific to BCR sequences or to other specific genes or groups of genes are also used. For example, each primer can contain a barcode and can also contain a 5’ overhang comprisinga 5’ overhang sequence (e.g., a 6-nucleotide sequence such as CACCTC, but any of a variety of overhang sequences can be used, including sequences of, e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length). Reverse transcriptase extends cDNA to form a cDNA / mRNA hybrid comprising the barcode (e.g., BC1 as shown in FIG. 2A), e.g., a well-specific barcode. The cells may then be pooled and split and redistributed into individual wells, and in subsequent steps, nucleic acid tags can be appended to the cDNA / mRNA hybrid via the 5’ overhang (FIG. 2B). The nucleic acid tags can contain a second barcode (e.g., BC2) and optionally a second DNA linker. The cells can be pooled and split and redistributed into individual wells. In some embodiments, a second nucleic acid tag can be ligated to the growing cDNA (see, e.g., FIG. 2C). In some embodiments, additional nucleic acid tags are ligated, e.g., with additional barcodes. Further any of the nucleic acid tags can comprise additional elements, e.g., next generation sequencing (NGS) adapter sequences (e.g., an Illumina adaptor sequence (shown as R2 in FIG. 2C) or a compatible equivalent thereof, primer binding sequences, other barcode or index sequences, random sequences, and / or capture elements such as a biotin molecule. In some embodiments, one or more of such elements are present in the final nucleic acid tag to be added to the (tagged) cDNA.

[0160] Suitable methods of combinatorial barcoding are described, e.g., in US Patent Nos. 10,900,065, 11,634,751, 11,168,355, 11,427,856, 11,555,216, 11,639,519, 11,680,283, 10,633,648, 11,421,221, US Pat. App. Pub. No. US 2021 / 0388415 Al, in Rosenberg et al., Science 360, 176-182 (2018), Rosenberg et al., BioRxiv (2017), “Scaling single cell transcriptomics through split pool barcoding,” doi.org / 10.1101 / 105163, Tran et al. BioRxiv (2022) “High sensitivity single cell RNA sequencing with split pool barcoding,” doi.org / 10.1101 / 2022.08.27.505512, the entire disclosures of all of which are herein incorporated by reference (including all supplemental material).

[0161] In some embodiments, combinatorial barcoding is accomplished by coupling the cDNA molecules generated in each cell (or nucleus) during reverse transcription (or, e.g., genomic DNA fragments or adapters appended to other molecules as described elsewhere herein, e.g., in the context of multi omics approaches) with a nucleic acid tag, wherein each nucleic acid tag comprises a barcode sequence, e.g., a well-specific barcode sequence or tag barcode. In particular embodiments, coupling the cDNA molecule with the nucleic tag comprises ligating the nucleic tag to the cDNA molecule (e.g., ligating the tag upon introducing the tag into the cell or nucleus, before another tag is added in a subsequent round, or alternatively adding one or more tags such that they are annealed but not ligated, and thenligating all of the annealed tags at a subsequent step, e.g., following lysis of the cells or nuclei). In some embodiments, each nucleic acid tag comprises a first strand comprising the barcode sequence, and further comprises a 3’ and / or 5’ region located 3’ and / or 5’ of the barcode. In some embodiments, the first strand comprises a 3' hybridization sequence extending from a 3' end of a labeling (i.e., barcode) sequence and / or a 5' hybridization sequence extending from a 5' end of the labeling (i.e., barcode) sequence. Each nucleic acid tag may also comprise a second (linker) strand including an overhang sequence. The overhang sequence may include (i) a first portion complementary to a 5' hybridization sequence of a different nucleic acid tag (e.g., a nucleic acid tag appended in a previous round of tagging) or to a 5' overhang sequence of an RT primer. The second (linker) strand may also comprise a sequence complementary to the 3' hybridization sequence of the first strand. In some embodiments, the first and second strands are preannealed before being added to the wells or aliquots containing the cells or nuclei.

[0162] In some embodiments, the nucleic acid tags comprise i) a tag barcode sequence, and ii) a 3’ hybridization sequence located 3’ of the barcode sequence and / or a 5’ hybridization sequence located 5’ of the barcode sequence, wherein multiple distinct tag barcode sequences are present among the nucleic acid tags used in the second plurality of aliquots, and wherein the tag barcode sequences present in each individual aliquot of the second plurality of aliquots are specific to the individual aliquot.

[0163] In some embodiments, the 3’ end of the nucleic acid tag is present within the 3’ hybridization sequence, and the 3’ end of the nucleic acid tag is brought into proximity of the 5’ end of the cDNA molecule by being preannealed to a linker nucleic acid strand that is complementary to the 3’ hybridization sequence of the nucleic acid tag and to the 5’ overhang sequence of the RT primer, or to the 3’ hybridization sequence of the nucleic acid tag and to the 5’ hybridization sequence of a previously coupled nucleic acid tag.

[0164] In some embodiments, the nucleic acid tags comprise a) a first strand comprising: i) a first strand barcode sequence; ii) a first strand 5’ hybridization sequence located 5’ of the first strand barcode sequence; and / or iii) a first strand 3’ hybridization sequence located 3’ of the first strand barcode sequence; and b) a second strand comprising: i) a second strand barcode sequence, wherein the second strand barcode sequence is complementary to the first strand barcode sequence; ii) a second strand hybridization sequence located 5’ of the second strand barcode sequence, wherein the second strand hybridization sequence is complementary to thefirst strand 3’ hybridization sequence; and / or iii) a second strand overhang sequence located 5’ of the second strand hybridization sequence; wherein the first strand and second strand are annealed such that the nucleic acid tag comprises: i) a double-stranded central region comprising the first strand barcode sequence annealed to the second strand barcode sequence and the first strand 3’ hybridization sequence annealed to the second strand hybridization sequence; and ii) a single-stranded overhang located at each end of the nucleic acid, wherein one of the two overhangs comprises the first strand 5’ hybridization sequence, and the other overhang comprises the second strand overhang sequence.

[0165] In some embodiments, the nucleic acid tags comprise a first or second strand, and / or a barcode sequence, hybridization sequence, or any of the elements or subsequences shown in any of Tables 7-11.

[0166] The barcode sequences present within the nucleic acid tags can be any of range of lengths, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In particular embodiments, the barcode sequences are 8 nucleotides in length. By varying 8 nucleotides, there are 65,536 possible unique sequences. In some embodiments, more than 8 nucleotides are used. In some other embodiments, fewer than 8 nucleotides are used. In particular embodiments, the first strand of the nucleic acid tag is preannealed to the second (linker) strand. In some embodiments, the linker strand includes sequence complementary to part of the RT primer or to a 5’ region in a previously coupled nucleic acid tag (i.e., appended to the cDNA in a previous round of tagging), thereby allowing it to hybridize and bring the 3' end of the barcodes into close proximity to the 5' end of the reverse transcription primer or the previously added tag. In some embodiments, the phosphate of the reverse transcription primeror previous tag can is ligated to the 3' end of the first-round barcodes by, e.g., T4 DNA ligase. A 5’ region of the nucleic acid tag (e.g., domain s2) can then provide an accessible binding domain for a linker oligo to be used in another round of barcoding. The nucleic acid tags (barcode oligos) can include a 5' phosphate that can allow ligation to the 3' end of another oligo by T4 DNA ligase.

[0167] In particular embodiments, the nucleic acid tags are ligated to the cDNAs (or adapter molecules, e.g., appended to genomic DNA fragments) during each round of labeling. In some embodiments, the methods of labeling nucleic acids in the first cell may comprise ligating at least two of the nucleic acid tags that are bound to the cDNAs (or genomic DNA fragments). In some embodiments, the nucleic acid tags are hybridized to the cDNAs (or adapters) during each round, and ligation is performed for all of the hybridized tags at a later stage, e.g., following cell lysis, i.e., ligation may be conducted before or after the lysing and / or the cDNA purification steps. Ligation can comprise covalently linking the 5' phosphate sequences on the nucleic acid tags to the 3' end of an adjacent strand or nucleic acid tag such that individual tags are formed into a continuous, or substantially continuous, barcode sequence that is bound to the 3' end of the cDNA or genomic DNA fragment sequence. In various embodiments, a double-stranded DNA or RNA ligase may be used with an additional linker strand that is configured to hold a nucleic acid tag together with an adjacent nucleic acid in a “nicked” double-stranded conformation. The double-stranded DNA or RNA ligase can then be used to seal the “nick.” In various other embodiments, a single-stranded DNA or RNA ligase may be used without an additional linker.

[0168] In some embodiments, following the ligation of the nucleic acid tags during each round, one or more unbound nucleic acid tags are removed (e.g., by washing the plurality of cells). For example, the methods may comprise removing a portion, a majority, or substantially all of the unbound nucleic acid tags. Unbound nucleic acid tags may be removed such that further rounds of the disclosed methods are not contaminated with one or more unbound nucleic acid tags from a previous round of a given method. In some embodiments, unbound nucleic acid tags may be removed via centrifugation. For example, the plurality of cells can be centrifuged such that a pellet of cells is formed at the bottom of a centrifuge tube. The supernatant (i.e., liquid containing the unbound nucleic acid tags) can be removed from the centrifuged cells. The cells may then be resuspended in a buffer (e.g., a fresh buffer that is free or substantially free of unbound nucleic acid tags).

[0169] In some embodiments, the plurality of cells (or nuclei) may be bound (or otherwise coupled or linked) to magnetic beads with affinity for the cells or nuclei, e.g., comprising or coated by an agent (such as Concanavalin A, or ConA, or an antibody) that comprises affinity for or is otherwise configured to bind the cell or nuclear membrane. When bound to such beads at any step of the herein-disclosed methods, the plurality of cells or nuclei can then, e.g., be pelleted using a magnet to draw them to one side of the reaction vessel so as to facilitate buffer exchanges and other manipulations carried out during subsequent steps of the protocol. In some embodiments, the plurality of cells may be placed in a cell strainer (e.g., a PLURISTRAINER® cell strainer) and washed with a wash buffer. For example, the plurality of cells may remain in the cell strainer while the wash buffer passes through the cell strainer. Wash buffer may include, e.g., a surfactant, a detergent, and / or about 5-60% formamide.

[0170] In some embodiments, the ligation can be stopped during each round of combinatorial labeling by adding an excess of oligo that is complementary to all or part of the linker (second) strand used during the same round of labeling. To stop each barcode ligation, oligo strands that are fully (or partially) complementary to the linker oligos can be added. These oligos can bind the linker strands attached to unligated barcodes and displace the unligated barcodes through a strand displacement reaction. The unligated barcodes can then be completely single-stranded. As T4 DNA ligase, for example, is unable to ligate single-stranded DNA to other single-stranded DNA , the ligation reaction will stop progressing. In particular embodiments, to ensure that all linker oligos are bound by the complementary (i.e., stop) oligos, a molar excess of the stop oligos (relative to the linker oligos) is added. In some embodiments, stop ligation strands are diluted into 10X Ligase Buffer and water, e.g., 264 pl stop ligation strand, 300 pl 10X T4 DNA Ligase Buffer, and 636 pl nuclease-free water.

[0171] As described above, in some embodiments, the nucleic acid tag (e.g., a final nucleic acid tag appended during the last round of multiple rounds of tagging) may comprise a capture agent such as, but not limited to, biotin, e.g., a 5' biotin. A cDNA labeled with a 5' biotincomprising nucleic acid tag may allow or permit the attachment or coupling of the cDNA to a streptavidin-coated magnetic bead, e.g., Cl beads. In some embodiments, a plurality of beads may be coated with a capture strand (i.e., a nucleic acid sequence) that is configured to hybridize to a final sequence overhang of a barcode. In some embodiments, cDNA may be purified or isolated by use of a commercially available kit (e.g., an RNEASY™ kit).

[0172] In some embodiments, one or more nucleic acid tags may comprise additional elements (in addition to biotin or another capture agent) such as a nucleotide sequence (e.g., comprising random and / or degenerate nucleotides) allowing the detection of PCR duplicates, primer binding sequences, adapter sequences for next-generation sequencing (NGS) (e.g., Illumina adapter sequences), and others. The random nucleotide sequences allow the computational removal of PCR duplicates, since these duplicates will have the same random sequence. In this way, each original transcript will only be counted once, even if multiple PCR duplicates are sequenced. Such sequences can contain any number of random successive nucleotides, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides or more, and can be used either alone or in conjunction with other sequence elements (such as other barcode sequences described herein, or fragment breakpoints) to allow the identification of PCR duplicates. The number of different random sequences per cell indicates how many unique RNA molecules can be detected per cell, which is directly related to how efficiently the molecules can be barcoded and processed to enable detection by next generation sequencing. In some embodiments, a plurality of distinct barcodes are used in each aliquot in order to allow the removal of PCR duplicates, as described elsewhere herein.Lysis

[0173] In some embodiments, the methods include lysing (i.e., breaking down the cell or nuclear structure) the plurality of cells (or nuclei) to release the tagged cDNA molecules from the plurality of cells or nuclei following combinatorial barcoding, thereby forming a lysate comprising the released tagged cDNA or genomic DNA fragment molecules. In some embodiments, the cells or nuclei comprising the tagged cDNAs (or other molecules) are divided into one or more samples or sublibraries, and the lysis is performed separately on each sample or sublibrary. In some embodiments, each sample or sublibrary can be tagged with one or more index or barcode sequences during a subsequent step in the herein- described methods (e.g., during an amplification step using unique dual indices, or UDIs, as described elsewhere herein). An exemplary overview of lysis and sublibrary generation are shown, e.g., in FIG. 2D.

[0174] The total number of sublibraries prepared can depend on various factors, including the number of cells in the plurality of cells or nuclei. For example, in some embodiments, the plurality of cells or nuclei comprises up to 10,000 cells or nuclei, and two sublibraries are prepared. In some embodiments, the plurality of cells or nucleicomprises up to 100,000 cells or nuclei, and 8 sublibraries are prepared. In some embodiments, the plurality of cells or nuclei comprises up to 1,000,000 cells or nuclei, and 16 sublibraries are prepared. Other numbers of cells or nuclei, including less than 10,000 and greater than 1,000,000 (e.g., 2 x 106, 3 x 106, 4 x 106, 5 x 106, 6 x 106, 7 x 106, 8 x 106, 9 x 106, 1 x 107, 2 x 107, 3 x 107, 4 x 107, 5 x 107, 6 x 107, 7 x 107, 8 x 107, 9 x 107, 1 x 108, or various numbers between 10,000 and 1,000,000, can be used, and a skilled artisan will be able to determine a suitable number of sublibraries.

[0175] Further, different numbers of cells can be added to each sublibrary as desired. Sublibraries with small cell numbers (e.g., 200-500) will be easier to sequence to saturation, and can serve, e.g., as a good quality control (QC) measure before sequencing additional sublibraries with much larger cell numbers. In some embodiments, the number of cells in each library can be, for example, 200, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, or more.

[0176] Once prepared, each sublibrary can be immediately processed, e.g., to prepare a sequencing library, or stored, e.g., at -80 °C. Further, while all of the sublibraries can be processed together, each sublibrary can be sequenced separately. Different numbers of cells can be added to each sublibrary as desired.

[0177] In some embodiments, the plurality of cells is lysed in a lysis solution (e.g., a solution comprising Tris-HCl, EDTA, NaCl, and SDS, e.g., 10 mM Tris-HCl (pH 7.9), 50 mM EDTA (pH 7.9), 0.2 M NaCl, 2.2% SDS) comprising an RNase inhibitor (e.g., 0.5 mg / ml ANTI-RNase, AMBION®) and a proteinase such as a serine protease, e.g., Proteinase K (e.g., 1000 mg / ml proteinase K (AMBION®)). The skilled artisan will be able to identify suitable conditions for cell (or nuclear) lysis. In some embodiments, lysis is performed at about 55 °C for about 3 hours with shaking (e.g., vigorous shaking). In some other embodiments, the plurality of cells is lysed using ultrasonication and / or by being passed through an 18-25 gauge syringe needle at least once. In yet some other embodiments, the plurality of cells is lysed by being heated to about 70-90 °C. For example, the plurality of cells may be lysed by being heated to about 70-90 °C. for about one or more hours. cDNA isolation

[0178] Following cell or nuclear lysis, the tagged cDNAs may be isolated from the lysed cells or nuclei. In some embodiments, RNase H (or another RNase) may be added to the cDNAto remove RNA. The methods may further comprise ligating at least two of the nucleic acid tags that are bound to the released cDNAs (e.g., in embodiments in which the tags were not ligated during each round of split-pool tagging). In some such embodiments, the methods may comprise ligating at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleic acid tags that are bound to the cDNAs. An exemplary overview of cDNA capture and amplification are shown, e.g., in FIGS. 3A-3C.

[0179] In some embodiments, the tagged cDNA molecules are isolated from the lysis solution with a purification or clean-up step, e.g. an SPRI bead cleanup, before binding the desired nucleic acids (i.e., cDNAs containing 5' biotin) to streptavidin beads. In particular embodiments, a protease inhibitor is added to lysates and then streptavidin beads are directly added (i.e., skipping the first SPRI isolation of nucleic acids). The protease inhibitor may include phenylmethanesulfonyl fluoride (PMSF), 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), a combination thereof, and / or another suitable protease inhibitor.

[0180] In some embodiments, the cDNA molecules are isolated using Streptavidin beads, e.g., Cl beads. For example, 20 pl of resuspended DYNABEADS® MYONE™ Streptavidin Cl beads (for each aliquot of cells) can be added to a 1.7 ml microcentrifuge tube (EPPENDORF®). The beads can be washed, e.g., 3 times, with, e.g., l x phosphate buffered saline Tween 20 (PBST) and resuspended in PBST (e.g., 20 pl PBST). In some embodiments, 900 pl PBST is added to the cell aliquot and 20 pl of washed Cl beads are added to the aliquot of lysed cells. The samples can, e.g., be placed on a gentle roller for 15 minutes at room temperature and then washed, e.g., 3 times with 800 pl PBST using a magnetic tube rack (EPPENDORF®). The beads can then be resuspended in, e.g., PBS such as 100 pl PBS.

[0181] In some embodiments, a microcentrifuge tube (EPPENDORF®) comprising a sample can be placed against a magnetic tube rack (EPPENDORF®) for, e.g., 2 minutes and then the liquid can be aspirated. In some embodiments, the beads can be resuspended in, e.g., an RNase solution (3 pl RNase Mix (ROCHE™), 1 pl RNase H (NEW ENGLAND BIOLABS®), 5 pl RNase H 10x Buffer (NEW ENGLAND BIOLABS®), and 41 pl nuclease- free water). The sample can be incubated under suitable conditions, e.g., at 37 °C for 1 hour, and then removed from the conditions and placed against a magnetic tube rack (EPPENDORF®) for, e.g., 2 minutes. The sample can be washed with, e.g., 750 pl of nuclease- free water+0.01% Tween 20 (H2O-T), without resuspending the beads and keeping the tube disposed against the magnetic tube rack. The liquid can then be aspirated. The sample can bewashed with 750 pl H2O-T without resuspending the beads and while keeping the tube disposed against the magnetic tube rack. Next, the liquid can be aspirated while keeping the tube disposed against the magnetic tube rack. The tube can then be removed from the magnetic tube rack and the sample can be resuspended in 40 pl of nuclease-free water.

[0182] FIGS. 3A-3B illustrate an exemplary embodiment of isolating cDNA molecules. After cell lysis, the biotinylated cDNA / mRNA hybrid binds to a streptavidin binder bead (FIG. 3 A). Molecules having biotin are collected and molecules lacking biotin are removed. Next, a template switch reaction is performed (FIG. 3B) using a template switching oligonucleotide (TSO). In some embodiments, the TSO comprises a template switching (TS) adapter comprising, e.g., a primer binding site (e.g., a template switching primer “TS primer”) to the 3’ end of the cDNA molecule for cDNA amplification.Second strand synthesis

[0183] In particular embodiments, to facilitate subsequent amplification, a common adapter sequence (or NGS adapter) is added to the 3 '-end of the released cDNA molecules following isolation of the released cDNA (i.e., cDNA / mRNA duplex). In particular embodiments, the common (or NGS) adapter sequence is the same, or substantially the same, for each of the cDNA molecules (i.e., within a given experiment). The addition of the common adapter may be conducted or performed in a solution including up to about 10% w / v of PEG, wherein the molecular weight of the PEG is between about 7,000 g / mol and 9,000 g / mol. In some embodiments, to prevent concatemers of the adapter oligo, dideoxy cytidine (ddC) can be included at the 3' end of the adapter oligo. In some embodiments, adapters are used with a phosphate at the 5' end and ddC at the 3' end. Several enzymes are capable of ligating singlestranded oligo to the 3 ' end of single-stranded DNA , e.g., T4 RNA ligase 1 (NEW ENGLAND BIOLABS®) or thermostable 5' AppDNA / RNA Ligase (NEW ENGLAND BIOLABS®).

[0184] In some embodiments, the adapter sequence is added to the 3 '-end of the released cDNA molecules by template switching (see, e.g., Picelli, S, et al. Nature Methods 10, 1096- 1098 (2013)). For example, template switching can be performed on the cDNA molecules, i.e., the cDNA / RNA duplexes attached to streptavidin beads. In some embodiments, up to 10% w / v PEG (molecular weight 7000-9000) is used in the template switch reaction. In some embodiments of the present methods, a TS primer sequence present within the adapter sequence introduced by the TSO is used for the amplification of tagged cDNA molecules, e.g., as illustrated in FIGS. 2A-2C.cDNA Amplification

[0185] In some embodiments, following the isolation of the tagged cDNA molecules and second strand synthesis, the cDNA molecules are amplified, e.g., amplified using WT primers (e.g., TSO and R2 primers) configured to broadly amplify all tagged cDNAs in the transcriptome.

[0186] In some embodiments, amplification comprises amplifying cDNA molecules using at least one pair of primers (e.g., whole transcriptome (WT) preamplification primers) configured to broadly amplify tagged cDNA molecules in the mixture. In some embodiments, the at least one pair of WT primers can comprise one primer complementary to an adapter sequence introduced by the TSO, and one primer complementary to an adapter sequence (e.g., R2 sequence) introduced by the last nucleic acid tag appended to the cDNA during split-pool labeling.

[0187] In some embodiments, amplification is performed using primers comprising a sequence shown as SEQ ID NO:713 or SEQ ID NO: 714 (Table 12), or a sequence comprising 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:713 or SEQ ID NO:714, or a sequence comprising not more than 1, 2, 3, 4, or 5 mismatches relative to SEQ ID NO:713 or SEQ ID NO:714.

[0188] In some embodiments, the amplification is performed by placing tubes comprising the (magnetic) beads with bound tagged cDNA molecules against a magnetic rack, removing and discarding the clear supernatant, and resuspending the beads in bind buffer, removing and discarding the supernatant, then removing the tubes from the magnetic rack and resuspending the beads in amplification reaction solution (e.g., a solution comprising an amplification master buffer, as well as general primers for amplifying the whole transcriptome). In some embodiments, primers specific to BCR sequences (e.g., BCR constant region sequences,including sequences specific to any one or more of IgM, IgG, IgD, IgA, and IgE isotypes, IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2 subclasses, BCR heavy chain, BCR light chain, BCR heavy chain constant region, BCR heavy chain variable region, BCR light chain constant region, BCR light chain variable region, BCR mu heavy chain, BCR delta heavy chain, BCR gamma heavy chain, BCR epsilon heavy chain, BCR alpha heavy chain, BCR kappa light chain, BCR lambda light chain, BCR complementarity-determining region (CDR), BCR CDR1, BCR CDR2, BCR CDR3, BCR CDR-H1, BCR CDR-H2, or BCR CDR-H3 domain) are also included, so as to further enrich the BCR sequences among the whole transcriptome. The resuspended beads can then be stored on ice (or a suitable temperature, e.g., at about 0, 1, 2, 3, 4, 5, 6, 7, or 8 °C). The tubes can then be placed in a thermocycler and subjected to suitable conditions for PCR amplification of the cDNAs. Following amplification, the tubes can be removed and stored, e.g., at 4 °C.

[0189] Following amplification of the cDNA molecules, the PCR products can be cleaned up, e.g., by the addition of solid phase reversible immobilization (SPRI) beads. SPRI beads are used to remove polynucleotides of less than about 200 base pairs, less than about 175 base pairs, or less than about 150 base pairs (see DeAngelis, M M, et al. Nucleic Acids Research (1995) 23(22):4742). In some embodiments, SPRI beads are used to remove polynucleotides of less than about 200 base pairs. The ratio of SPRI bead solution to amplified cDNA molecule solution may be between about 0.9:1 and about 0.7: 1, between about 0.875 : 1 and about 0.775 : 1 , between about 0.85: 1 and about 0.75: 1, between about 0.825: 1 and about 0.725: 1, about 0.8:1, or another suitable ratio.

[0190] In some embodiments, the SPRI bead solution may include between about 1 M and 4 M NaCl, between about 2 M and 3 M NaCl, between about 2.25 M and 2.75 M NaCl, about 2.5 M NaCl, or another suitable amount of NaCl. The SPRI bead solution may also include between about 15% w / v and 25% w / v polyethylene glycol (PEG), wherein the molecular weight of the PEG is between about 7,000 g / mol and 9,000 g / mol (PEG 8000). In various embodiments, the SPRI bead solution may include between about 17% w / v and 23% w / v PEG 8000, between about 18% w / v and 22% w / v PEG 8000, between about 19% w / v and 21% w / v PEG 8000, about 20% w / v PEG 8000, or another suitable % w / v PEG 8000.

[0191] In one embodiment, 20 pl of the RNase-treated beads can be added to a single PCR tube. 80 pl of ligase mix (5 pl T4 RNt Ligase 1 (NEW ENGLAND BIOLABS®), 10 pl 1 OX T4 RNA ligase buffer, 5 pl BC_0047 oligo at 50 pM, 50 pl 50% PEG 8000, and 10 pl 10 mMATP) can be added to the 20 pl of beads in the PCR tube. 50 pl of the ligase mixed with the beads can be transferred into a new PCR tube to prevent too many beads from settling to the bottom of a single tube and the sample can be incubated at 25 °C for 16 hours.Preparation of sequencing libraries

[0192] Following the cDNA amplification, two sequencing libraries are prepared for each cDNA sublibrary: a Whole Transcriptome (WT) Library, and a BCR-specific library. In embodiments in which, e.g., 8 sublibraries have been prepared prior to lysis, this will result, e.g., in a total of 16 sequencing libraries from the previous 8 sublibraries made. In particular embodiments, during preparation of the WT and BCR library preparation, an additional (e.g., fourth) sublibrary-specific barcode can be added to the cDNA. In particular embodiments, this additional (e.g., fourth) barcode is an Illumina Unique Dual Index (UDI).Whole transcriptome (WT) sequencing libraries

[0193] In some embodiments, to prepare WT sequencing libraries, the amplified WT cDNA molecules are fragmented, an adapter comprising, inter alia, a primer binding sequence is appended to the fragmented ends, and an additional amplification reaction is performed to introduce one or more index sequences (e.g., unique dual indexes, or UDIs) and sequencing primer binding sites for NGS sequencing (see, e.g., FIG. 7). The WT cDNAs can be fragmented, e.g., using a fragmentation enzyme and fragmentation buffer. In some embodiments, the amplified cDNA molecules are fragmented by incubating with the fragmentation enzyme and buffer at 32 °C for 10 minutes and are then held at 65 °C for, e.g., 30 minutes. In some embodiments, following fragmentation of the DNA, the fragment ends are repaired and A-tailed, and the adapter is ligated to the ends. For example, in some embodiments, an Illumina Truseq R1 Adapter is ligated to the 5’ end of the DNA. In some embodiments, the ligation of the adapter to the ends of the fragments of the amplified cDNA can be preceded and / or followed by an SPRI clean-up step, e.g., using Ampure XP or KAPA Pure Beads.

[0194] In some embodiments, the cleaned-up molecules are then subjected to an additional round of amplification, e.g., adding P5 / P7 adapter sequences. A fourth barcode can also be added. In some embodiments, the additional barcodes correspond to unique dual indexes (UDI), e.g., with different well-specific index primers used for each sublibrary (see, e.g., Table 36). In some embodiments, the indexing round of amplificationcan be preceded and / or followed by an additional SPRI clean up step (e.g., using Ampure XP or KAPA Pure Beads).

[0195] FIGS. 4A-4C illustrate an exemplary embodiment of preparation of whole transcriptome libraries for sequencing. In this embodiment, sublibrary cDNA is fragmented to a size compatible with a suitable sequencing platform (FIG. 4A), e.g., Illumina sequencing or other compatible sequencing platforms. A second adaptor (e.g., an R1 adapter) is then ligated to the fragmented ends of the cDNA (FIG. 4B). Lastly, a final PCR (FIG. 4C) amplifies the fragmented cDNA and appends, e.g., UDIs and / or P5 and P7 adaptors.BCR Libraries

[0196] In some embodiments, for the preparation of BCR cDNA libraries, specific cDNAs (e.g., BCR-encoding cDNAs) are enriched by hybridization-based methods, e.g., by gene capture using a BCR-specific panel. For example, a panel can be used (e.g., a Human BCR panel, Mouse BCR panel, or chimeric human-mouse BCR panel), comprising probes specific to human and / or mouse BCR sequences, e.g., BCR sequences encoding any one or more of BCR IgM, IgG, IgD, IgA, and IgE isotypes, IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2 subclasses, BCR heavy chain, BCR light chain, BCR heavy chain constant region, BCR heavy chain variable region, BCR light chain constant region, BCR light chain variable region, BCR mu heavy chain, BCR delta heavy chain, BCR gamma heavy chain, BCR epsilon heavy chain, BCR alpha heavy chain, BCR kappa light chain, BCR lambda light chain, BCR complementarity-determining region (CDR), BCR CDR1, BCR CDR2, BCR CDR3, BCR CDR-H1, BCR CDR-H2, or BCR CDR-H3 domain. FIGS. 5A-5B illustrate an exemplary embodiment of hybrid capture to enrich for BCR-specific sequences among the amplified cDNAs, and FIGS. 6A-6B illustrate an exemplary embodiment of the amplification of captured BCR sequences.

[0197] In some embodiments, the probes correspond to a portion of the potential BCR encoding sequences, e.g., to heavy or to light chains, to IgA, IgG, IgM, IgE, or IgD heavy chains, to kappa or lambda light chains, to variable regions, to constant regions, to CDR1, CDR2, or CDR3 regions, to intronic regions or exonic regions, etc., or any combination of any one or more of any of these elements. In some embodiments, the probes collectively cover the all of the potential BCR encoding sequences in the cell, e.g., at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the potential BCR coding sequences (orBCR transcripts) in the cell. In some embodiments, the probes provide uniform coverage of the potential BCR encoding sequences or transcripts. In some embodiments, the probes provide enhanced coverage of certain BCR-encoding sequences or regions (e.g., variable regions).

[0198] In some embodiments, the panel comprises from 100-1000 BCR probes, or at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 BCR probes. The probes can be, e.g., from 20-500 nucleotides in length, or at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 nucleotides in length. In some embodiments, the probes are all or on average about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 nucleotides in length.

[0199] In some embodiments, the probes comprise homology or complementarity to any one or more of the sequences shown in Table 13 or 14, or disclosed herein as SEQ ID NOS: 750-1121, or to a subsequence of any of the sequences shown in Table 13 or 14 or disclosed herein as SEQ ID NOS: 750-1121, or comprise at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology or complementarity to any of the sequences shown in Table 13 or 14 or disclosed herein as SEQ ID NOS: 750-1121.

[0200] In some embodiments, the B cells comprise human B cells, and the probes comprise homology or complementarity to any one or more of the sequences shown in Table13 or disclosed herein as SEQ ID NOS: 750-944, or to a subsequence of any one or more of the sequences shown in Table 13 or disclosed herein as SEQ ID NOS: 750-944, or comprise at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology or complementarity to any of the sequences shown in Table 13 or disclosed herein as SEQ ID NOS: 750-944, or a subsequence thereof.

[0201] In some embodiments, the B cells comprise mouse B cells, and the probes comprise homology or complementarity to any one or more of the sequences shown in Table14 or disclosed herein as SEQ ID NOS: 945-1121, or to a subsequence of any one or more of the sequences shown in Table 14 or disclosed herein as SEQ ID NOS: 945-1121, or comprise at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology or complementarity to any of the sequences shown in Table 14 or disclosed herein as SEQ ID NOS: 945-1121, or a subsequence thereof.

[0202] In some embodiments, the B cells comprise chimeric B cells (e.g. express transgenic BCRs comprising, e.g., portions of human and portions of mouse BCR sequences,such as mouse constant sequences and mouse variable sequences), and probes are used that target sequences in both Table 13 and Table 14, e.g., using probes that comprise homology or complementarity to any one or more of the sequences shown in Table 13 or as SEQ ID NOS: 750-944 and to one or more of the sequences shown in Table 14 or as SEQ ID NOS: 945-1121, or that comprise homology or complementarity to a subsequence or sequence of any one or more of the sequences shown in Table 13 or as SEQ ID NOS: 750-944 and to a subsequence or sequence of one or more of the sequences shown in Table 14 or as SEQ ID NOS: 945-1121, or that comprise at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology or complementarity to any of the sequences shown in Table 13 or as SEQ ID NOS: 750-944 and to a sequence shown in Table 14 or as SEQ ID NOS: 945-1121.

[0203] Exemplary BCR target genes include, but are not limited to, IGHA1, IGHA2, IGHD, IGHD1-1, IGHD1-7, IGHD1-14, IGHD1-20, IGHD1-26, IGHD1OR15-1A, IGHD1OR15-1B, IGHD2-2, IGHD2-8, IGHD2-15, IGHD2-21, IGHD2OR15-2A,IGHD2OR15-2B, IGHD3-3, IGHD3-9, IGHD3-10, IGHD3-16, IGHD3-22, IGHD3OR15-3A, IGHD3OR15-3B, IGHD4-4, IGHD4-11, IGHD4-17, IGHD4-23, IGHD4OR15-4A,IGHD4OR15-4B, IGHD5-5, IGHD5-12, IGHD5-18, IGHD5-24, IGHD5OR15-5A,IGHD5OR15-5B, IGHD6-6, IGHD6-13, IGHD6-19, IGHD6-25, IGHD7-27, IGHE, IGHG1, IGHG2, IGHG3, IGHG4, IGHGP, IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, IGHJ6, IGHM, IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18, IGHV1-24, IGHV1-38-4, IGHV1-45, IGHV1-46, IGHV1-58, IGHV1-69, IGHV1-69-2, IGHV1-69D, IGHV1OR15-1, IGHV1OR15-5, IGHV1OR15-9, IGHV1OR21-1, IGHV2-5, IGHV2-26, IGHV2-70, IGHV2-70D, IGHV2OR16-5, IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-16, IGHV3- 20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-30-3, IGHV3-30-5, IGHV3-33, IGHV3-35, IGHV3-38, IGHV3-38-3, IGHV3-43, IGHV3-43D, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-64D, IGHV3-66, IGHV3-72, IGHV3-73, IGHV3-74, IGHV3OR16-8, IGHV3OR16-9, IGHV3OR16-10, IGHV3OR16-12, IGHV3OR16-13, IGHV3OR16-17, IGHV4-4, IGHV4-28, IGHV4-30-1, IGHV4-30-2, IGHV4-30-4, IGHV4-31, IGHV4-34, IGHV4-38-2, IGHV4-39, IGHV4-59, IGHV4-61, IGHV4OR15-8, IGHV5-10-1, IGHV5-51, IGHV6-1, IGHV7-4-1, IGHV7-81, IGHV8-51-1, IGKC, IGKJ1, IGKJ2, IGKJ3, IGKJ4, IGKJ5, IGKV1-5, IGKV1-6, IGKV1-8, IGKV1-9, IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37, IGKV1-39, IGKV1D-8, IGKV1D-12, IGKV1D-13, IGKV1D-16, IGKV1D-17, IGKV1D-33, IGKV1D-37, IGKV1D-39, IGKV1D- 42, IGKV1D-43, IGKV1OR2-0, IGKV1OR2-108, IGKV2-24, IGKV2-28, IGKV2-29,IGKV2-30, IGKV2-40, IGKV2D-24, IGKV2D-26, IGKV2D-28, IGKV2D-29, IGKV2D-30, IGKV2D-40, IGKV3-7, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-7, IGKV3D-11, IGKV3D-15, IGKV3D-20, IGKV3OR2-268, IGKV4-1, IGKV5-2, IGKV6-21, IGKV6D-21, IGKV6D-41, IGLC1, IGLC2, IGLC3, IGLC6, IGLC7, IGLJ1, IGLJ2, IGLJ3, IGLJ4, IGLJ5, IGLJ6, IGLJ7, IGLV1-36, IGLV1-40, IGLV1-44, IGLV1-47, IGLV1-50, IGLV1-51, IGLV2- 8, IGLV2-11, IGLV2-14, IGLV2-18, IGLV2-23, IGLV2-33, IGLV3-1, IGLV3-9, IGLV3-10, IGLV3-12, IGLV3-16, IGLV3-19, IGLV3-21, IGLV3-22, IGLV3-25, IGLV3-27, IGLV3-32, IGLV4-3.

[0204] The probes can comprise deoxyribonucleotides, ribonucleotides, or analogs, derivatives, or otherwise modified forms of deoxyribonucleotides or ribonucleotides, or combinations of any of these components. For example, the probes can comprise analogs or modifications such as PNA (peptide nucleic acid), LNA (locked nucleic acid), UNA (unlocked nucleic acid), triazole-linked DNA, 2'-fluoro-RNA, 2'-O-methyl RNA, phosphorothioate, 2'- O-(2-methoxyethyl)-RNA (MOE), phosphorodiamidate morpholinos (PMO), 5-nitroindole, 5- Methyl dC (5-Me dC), 2’-O-methoxy-ethyl (2’-M0E), or other nucleotides or nucleotide analogs.Ill

[0205] In some embodiments, one or more sublibraries can be pooled (e.g., up to 8 WT sublibraries) for the hybrid capture reaction. In some embodiments, the hybridization step (i.e., in which the tagged cDNA molecules are incubated with the BCR panel) is preceded by a blocking step, in which blockers are added to the library pool to reduce off-target binding during hybridization. For example, in some embodiments, the transcripts are first blocked with Evercode library-specific and repetitive sequences blockers.

[0206] In some embodiments, the probes (e.g., the BCR-specific probes) comprise a capture agent such as biotin, such as to allow the capture of bound cDNAs following hybridization. Hybridization conditions can depend on various factors, e.g., the length and / or G / C content of the probes, the number of targeted sequences and potential off-target sequences, the buffer used, etc., and can be optimized to identify conditions allowing the probes of the panel to bind optimally to the BCR sequences among the tagged cDNA molecules, with a minimal or acceptable amount of non-specific binding. An exemplary protocol for performing hybrid capture is described, e.g., in Example 1. In some embodiments, the library pool is first denatured in the presence of blockers by heating, e.g., to 95 °C (e.g., for 5 minutes), before being placed at room temperature to cool. Separately, the probes are heated to 95 °C (e.g., for 2 minutes) and then also allowed to cool at room temperature, before mixing the probes and the blocked library pool and incubating, e.g., at 70 °C. In some embodiments, the hybridization reaction is performed at, e.g., about 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, 65 °C, 66 °C, 67 °C, 68 °C, 69 °C, 70 °C, 61 °C, 72 °C, 73 °C, 74 °C, or 75 °C. In some embodiments, the hybridization reaction is performed for at least about 5 hours, or at least about 10 hours, or for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours. In some embodiments, the hybridization reaction is performed at 70 °C for 15-17 hours.

[0207] Following hybridization, the hybridized BCR transcripts are captured and then amplified (see, e.g., FIGS. 6A-6B). First, the tagged BCR cDNA molecules bound to a probe in the panel are isolated, e.g., using streptavidin beads (which bind to the biotin present within the probes) and washed, e.g., to remove unbound sequences from the library. In some embodiments, the mixture is kept constantly at 70 °C, e.g., from the hybridization step through transfer to streptavidin beads, in order to minimize off-target binding. In some embodiments, the bound beads are transferred to a new tube, e.g., to reduce background from non-specific binding to the surface of the original tube. The bound beads can then be washed, e.g., washed three times to remove unbound sequences from the library, and the beads ultimately removed from the magnet and resuspended, e.g., in nuclease-free water.

[0208] The captured BCR sequences are then amplified for further enrichment prior to sequencing. For example, the library pool can be subjected to PCR, with the number of cycles determined by the amount of nucleic acids present in the library pool (see, e.g., Table 38), e.g., using exemplary conditions shown in Table 39. In some embodiments, an SPRI clean-up step is performed immediately following PCR, e.g., using 1.8X SPRI beads. In some embodiments, the concentration and / or size distribution of the BCR library is assessed using, e.g., a Qubit dsDNA HS (High Sensitivity) Assay kit. In some embodiments, the size distribution is compared to a reference distribution (e.g., as shown in FIGS. 11 A-l IB), which can be of use in determining the suitability of the library for sequencing, e.g., using parameters and conditions as described elsewhere herein and as known in the art. The amplified double stranded libraries are ready to be sequenced.Sequencing and analysis

[0209] BCR sequencing libraries can be sequenced together or separately from Whole Transcriptome libraries. In particular embodiments, sequencing reads from both libraries (i.e., BCR and WT libraries) are grouped by cell barcodes (e.g., RT primer barcodes, nucleic acid tag barcodes, UDI barcodes, and combinations thereof). Each barcode combination should correspond to the cDNA from a single cell. In particular embodiments, only reads with valid barcodes are retained. For the WT library, the sequencing reads with each barcode combination can be aligned to a reference genome, e.g., to a reference human genome. Multiple reads with the same random identifier sequence are counted as a single read. In some embodiments, reads with random identifier sequences with two or less mismatches are assumed to be generated by sequencing errors and are counted as a single read.

[0210] Sequence reads from each library (i.e., the whole transcriptome library and the BCR library) comprising the same barcode sequence combinations are then associated to correlate the expression of, e.g., a given BCR isotype or clonotype with the whole transcriptome in the same cell or nucleus.

[0211] In some embodiments, the sequence reads are analyzed using a software platform such as Trailmaker™, which can process raw sequencing data (e.g., FASTQ files) to generate, e.g., quality control reports, count matrices, and log files, and can also provide various tools to, e.g., visualize, assess, and annotate aspects of the data such as cell types, gene expression, clustering, trajectory analysis, and others.Kits

[0212] Another aspect of the disclosure relates to kits for labeling nucleic acids within B cells or nuclei derived therefrom. In some embodiments, the kit may comprise at least one reverse transcription primer comprising a 5' overhang sequence, and / or a poly(T) sequence or a random nucleotide sequence, such as one or more RT primers as shown in any of Tables 1- 6, or comprising one or more element or subsequence shown in any of the sequences of any one or more of Tables 1-6. In some embodiments, the kits may include a plurality of nucleic acid tags, e.g., one or more sets of nucleic acid tags, such as one or more nucleic acid tags, or sets of nucleic acid tags, as shown in any one or more of Tables 7-11, or comprising an element or subsequence shown in any of the sequences of Tables 7-11.

[0213] In some embodiments, the kit may comprise one or more BCR-specific probes for performing hybrid capture of BCR-encoding transcripts, e.g., according to the herein-described methods. In some embodiments, the kit comprises one or more probes targeting a sequence shown Tables 13 and / or 14, or a subsequence thereof. In some embodiments, the kit comprises additional reagents useful to hybrid capture, such as blockers, buffers, reaction vessels, etc.

[0214] In various embodiments, the kit may further comprise at least one of a reverse transcriptase, a fixation agent, a permeabilization agent, a ligation agent, and / or a lysis agent.

[0215] Another aspect of the disclosure relates to kits for labeling other molecules within the B cells, or nuclei derived therefrom. For example, the kits as disclosed above may include one or more reagents or elements for multiomics applications, e.g., to label gDNA or one or more proteins, peptides, or antigens within the B cells, or nuclei derived therefrom, in addition to RNA corresponding to BCR genes and whole transcriptome.

[0216] In some embodiments, the kit comprises one or more reaction vessels or containers for the any one or more of the herein-described compositions or methods. For example, in particular embodiments the kit comprises one or more multi-well plates such as 96-well plates. In some embodiments, the kit comprises one or more multi-well plates pre-loaded with barcoded RT primers, with nucleic acid tags, or with indexed primers (e.g. UDI primers) according to the present disclosure. In some embodiments, the kit comprises amplification primers comprising an index sequence, e.g., a sample index sequence. In particular embodiments, the primers comprise dual UDIs, e.g., comprising one or more sequences shown in Table 37.EXAMPLES

[0217] The following examples are illustrative of disclosed methods and compositions. In light of this disclosure, those of skill in the art will recognize that variations of these examples and other examples of the disclosed methods and compositions would be possible without undue experimentation.Example 1. Exemplary protocol for the preparation of WT and BCR sequencing libraries from B cells (or B cell nuclei)Workflow

[0218] Evercode split-pool combinatorial barcoding brings a simple workflow to large- scale single cell RNA-seq experiments. The Evercode Human BCR and Whole Transcriptome kit can profile, e.g., up to 100,000 cells across up to 48 different biological samples or experimental conditions. The Evercode BCR Mini and Whole Transcriptome kit can profile, e.g., up to 10,000 cells across up to 12 different biological samples or experimental conditions, and the Evercode BCR Mega and Whole Transcriptome kit can profile, e.g., up to 1,000,000 cells across up to 96 different biological samples or experimental conditions.

[0219] Evercode fixation kits fix and permeabilize cells so they act as individual reaction compartments. This eliminates the need for dedicated microfluidics instrumentation. Through four rounds of barcoding, the transcriptome of each fixed cell is uniquely labeled. The four rounds of barcoding can yield a vast number of possible barcode combinations, which is more than sufficient to uniquely label up to 100,000 (or 10,000, or 1,000,000) cells while avoiding doublets. After sequencing, the Parse Biosciences Analysis Pipeline assigns reads that share the same four barcode combinations to a single cell. The Evercode BCR and Whole Transcriptome kit is a hybridization-based technology that captures B cells' full length V(D)J sequences. FIGS. 1-6 and Table 15 provide a high-level overview of the workflow.Sample Input

[0220] When working with human B cells, the protocol begins with cells fixed with an Evercode Cell Fixation v3 kit. It is also compatible with samples fixed with Evercode Cell Fixation v2. When working with mouse B cells, use the Evercode Mouse TCR / BCR Cell Fixation workflow, which includes a murine RNase Inhibitor.

[0221] Even if samples were counted before freezing, we strongly recommend counting cells / nuclei again after thawing to account for any changes during storage and freeze thaw. Typically, a 5-15% decrease after thawing should be expected. These counts are used to determine how cells / nuclei are loaded in the Round 1 Plate, and their accuracy is critical to recover the desired number of cells.

[0222] When processing many fixed samples, we recommend aliquoting samples after fixation and counting the aliquots the day before using an Evercode kit. The Evercode Fixation User Manuals outline recommendations for generating aliquots. Because aliquots have undergone a similar storage time and a freeze thaw, cell counts from these aliquots will be more representative than using counts from immediately after fixation. Aliquots should be thawed in a water bath set to 37°C in sets of 2-4 and counted with a hemocytometer or alternative counting device. Counts should be recorded in the Sample Loading Table, and any remaining counting aliquot should be discarded. Once fixed samples have been thawed, they should not be refrozen.Cell / Nuclei Counting and Quality Assessment

[0223] We recommend a hemocytometer for counting, but alternative counting devices can also be used. If possible, validate counts from alternative devices to a hemocytometer when using Evercode Whole Transcriptome kits for the first time. When first using Evercode kits, we suggest saving images at each counting step. To assess sample quality, we recommend using viability stains like trypan blue or acridine orange and propidium iodide (AO / PI). After fixation, the cells are permeabilized and will appear dead with viability stains.

[0224] Examples of trypan blue stained fixed cells are shown, e.g., in FIGS. 9A-9C. High quality fixed samples have single distinct cells with <5% cell aggregation and no debris. Higher levels of aggregation will lead to elevated doublets after sequencing. Whenquantifying fixed samples, it is critical to avoid counting cell debris to avoid overestimating the number of cells.Avoiding RNase Contamination

[0225] Take standard precautions to avoid introducing RNases into samples or reagents throughout the workflow. Always wear proper laboratory gloves and use aseptic technique. Although RNases are not inactivated by ethanol or isopropanol, they are inactivated by products such as RNaseZap RNase Decontamination Solution (Thermo Fisher Scientific). These can be sprayed on benchtops and pipettes. Filtered pipette tips should be used to reduce RNase contamination from pipettes.Centrifugation

[0226] A range of centrifugation speeds and durations are provided in this protocol. Optimize centrifugation conditions for each sample type to balance retention and resuspension efficiencies. Use a swinging bucket rotor for all high-speed centrifugation steps in this protocol. A fixed-angle rotor will lead to substantial cell loss.Optimizing Cell Recovery

[0227] It is critical to thoroughly resuspend the cells after centrifugation throughout the protocol. Resuspend by slowly and repeatedly pipetting up and down until no clumps are visible. Due to cell adherence to tubes, carefully pipette up and down along the bottom and sides of tubes to minimize cell loss.

[0228] We do not recommend wide bore pipette tips as they make it difficult to resuspend cell pellets adequately. Ensure that the 15 mL centrifuge tubes that will be used are polypropylene, as polystyrene tubes will lead to substantial sample loss. The first time using an Evercode kit, we recommend retaining supernatants after each barcoding step. In the unlikely event of unexpectedly high sample loss, these supernatants can be analyzed to identify points for optimization.Cell Strainers

[0229] A cell strainer with an appropriately sized mesh should be used throughout the protocol. Although 30-40 pm is appropriate for many cell types, the mesh size should be chosen based on the sample type. To maximize cell retention with cell strainers, press thepipette tip directly against the mesh. Ensure ample pressure is applied to hold contact between the tip and the strainer to force liquid through in ~1 second.Vortexing

[0230] Unless specified in an individual step, we strongly discourage vortexing samples or enzymes throughout the protocol.Plate Sealing

[0231] While sealing or unsealing 96 well plates, do not splash liquid onto the PCR plate seal or between wells. Securing plates in PCR tube racks will minimize this occurrence. PCR plate seals may be difficult to remove. Carefully peel the PCR plate seal while applying downward pressure on the plate to keep it in the PCR tube rack.Magnetic Racks and Bead Cleanups

[0232] The Parse Biosciences Magnetic Rack (Parse Biosciences) uses powerful rare earth magnets for rapid and efficient magnetic bead purifications for 0.2 mL tubes. The rack has high and low magnet positions important for optimal yield at key steps. To alternate between the positions, the rack can be flipped upside down so the magnet is closer to the top (high) or bottom (low) of the 0.2 mL tubes.

[0233] To ensure material is not lost during bead purifications, ensure supernatants are completely clear before moving to the next step. The incubation times at each step are recommendations, but visual confirmation of clearing should be used to make the final determination. Discarding any beads in supernatants will result in a reduction of transcripts and genes detected per cell.Sub library Loading

[0234] The Evercode BCR kit generates 8 sublibraries with distinct Illumina indexing barcodes, which can be processed and sequenced independently or concurrently after Section 1 of the protocol. The number of cells or nuclei per sublibrary is determined when the cells are divided into sublibraries in Section 1.5. When working with a new sample type, it can be beneficial to process a single sublibrary to optimize PCR conditions before running the remaining sublibraries. Sublibraries can be loaded with different numbers of cells, and the maximum number of cells that can be analyzed is the sum of cells / nuclei across all sublibraries. Asymmetric sublibrary loading can enable cost-effectivesequencing quality control. One sublibrary can be loaded with a few hundred cells / nuclei and sequenced very deeply. These data can be used to choose an appropriate sequence depth for the remaining sublibraries. Sublibraries generated in Section 4 can also be concentrated with vacuum centrifugation rather than SPRI bead concentration.Indexing Primers

[0235] The UDI Plate - WT is a 96-well plate containing 48 unique dual indexing (UDI) primers. Each well is a single-use reaction sufficient for one Barcoding Round 4 PCR reaction for a single sublibrary. Thus, the UDI Plate - WT can be used for multiple Evercode Whole Transcriptome kits. The UDI Plate - WT is sealed with a pierceable foil to minimize cross-contamination. The plate seal should be wiped with 70% ethanol and pierced with a new pipette tip immediately prior to use. Avoid splashing or mixing the liquid between individual wells. Once a well has been used, it should not be resealed or reused. We recommend selecting UDIs column-wise from left to right (starting with indices 1-8). UDI sequences can be found in Table 37.BCR Library Generation

[0236] The BCR libraries generation protocol begins with indexed sublibraries obtained with the Evercode BCR kit. For each library pool, up to 8 indexed sublibraries can be used for BCR library preparation (or, for BCR Mini Kits, up to 2 indexed sublibraries can be used, or for BCR Mega kits up to 16 sublibraries can be used). Pooling sublibraries requires at least a concentration of 5 ng / pL per sublibrary. We recommend adding a minimum of 100 ng of each sublibrary to the BCR library pool for a maximum of 800 ng of total input. To ensure even sequencing coverage, sublibraries with similar cell numbers should be mixed in equal proportion. However, if any sublibraries have less than 100 ng of input, sublibraries can be pooled in equal proportion to a total of 400 ng. Adjust the number of PCR cycles based on the indexed sublibraries input amount to ensure optimal amplification.

[0237] Tables 16 and 17 provide a list of components used in the exemplary protocol.Section 1: In situ Cell Barcoding1.1. Set up and Sample Counting

[0238] Prior to barcoding, cells are thawed and counted. Appropriate dilutions, loading concentrations, and loading positions are determined. To set up for barcoding: 1. Cool a centrifuge with swinging bucket rotors to 4°C. 2. Set a water bath to 37°C. 3. Fill a bucket with ice. 4. Prepare a hemocytometer, flow cytometer, or other cell counting device. 5. Thaw the previously fixed cell / nuclei samples in a water bath set to 37°C until all ice crystals dissolve. 6. Thoroughly mix each sample by pipetting and store on ice. 7. While minimizing time on ice, count the number of cells in the sample with a hemocytometer or alternative cell counting device. Record the cell count.

[0239] When processing many fixed samples, we recommend aliquoting samples after fixation and counting the aliquots the day before using an Evercode Whole Transcriptome kit. Record the sample names and cell count. Place the Round 1 Plate into a thermocycler and run the program shown in Table 15. Dilute each sample with sample dilution buffer and store on ice. Proceed immediately to Section 1.2.1.2 Barcoding Round 1

[0240] Samples are loaded into Round 1 Plate. An in situ reverse transcription reaction adds well-specific barcodes that also serve as sample barcodes. Cells are then pooled, centrifuged, and resuspended. To add round 1 barcodes: 1. Gently remove the Round 1 Plate from the thermocycler, place in a 0.2 mL tube rack, and centrifuge for 1 minute at100 x g at 4°C. 2. Remove the Round 1 Plate from the centrifuge, place in a PCR tube rack, remove the plate seal, and store on ice. 3. With the Round 1 Plate on ice, add 14 pL of each diluted sample to the appropriate wells of Round 1 Plate. Mix immediately after dispensing each sample by pipetting 3x. Note: When pipetting the same sample into many wells, the sample must be mixed by gentle pipetting prior to each transfer to avoid cell settling. Do not vortex the samples. 4. While secured in a PCR tube rack on a flat surface, add a new plate seal. 5. Place the Round 1 Plate into a thermocycler and run the program shown in Table 19. Upon completion, proceed immediately to the next step.

[0241] 6 Remove the Round 1 Plate from the thermocycler, place in a PCR tube rack, and store on ice. 7. Place the Round 2 Plate into a thermocycler and run the program shown in Table 20. 8. While secured in a PCR tube rack on a flat surface, remove the plate seal from the Round 1 Plate. 9. With the plate and tube on ice, pool all wells from the Round 1 plate into a 15 mL centrifuge tube and pipette as follows: i. With a multichannel P200 set to 30 pL, mix the sample in row B of a 96-well plate by pipetting 3x. ii. Transfer 30 pL from row B to row A of the 96-well plate, iii. Repeat i-ii for rows C-D to mix the sample, then transfer to row A. iv. Transfer any residual liquid in rows B-D to row A with a multichannel P20 set to 10 pL. v. Ensure the cells in row A are in suspension as describedin i. Then, transfer the total volume of each well in row A into the same 15 mL tube with a single channel P200 set to 200 pL.

[0242] 10. Add 9.6 pL of spin additive to the 15 mL tube with pooled cells. Do not discard the spin additive as it will be needed in another step. 11. Mix by gently inverting the tube just once. 12. Centrifuge the 15 mL tube in a swinging bucket rotor for 10 minutes at 200-500 x g at 4°C. Immediately move to the next step after centrifugation. Note: Ideal centrifugation speed and duration should be determined empirically for each sample type to optimize retention and resuspension efficiencies. Overly aggressive centrifugation can damage cell integrity and decrease data quality. If the centrifugation speeds used during fixation gave satisfactory retention, they should be used throughout this protocol. Move quickly and handle the samples gently to avoid dislodging the pellet, which will have a significant impact on data quality.

[0243] 13. Remove the supernatant until about ~40 pL of liquid remains above the pellet. Use a Pl 000 for the first 1 mL and then a P200 for the remaining volume. Note: Depending on the number of cells and cell types, a pellet may or may not be visible. 14. Fully but gently resuspend the pellet in 1 mL of resuspension buffer. 15. Add an additional 1 mL of resuspension buffer for a total addition of 2 mL. Store on ice. 16. Proceed immediately to section 1.3 (Round 2 Barcoding).

[0244] Note: If the low input fixation workflow was performed (as described, e.g., in Example 3), execute the following step before proceeding to Section 1.3: 17. Pipette the sample through a cell strainer into a new 15 mL tube with a P1000. Before transfer, gently mix the cells by pipetting 2x.1.3 Barcoding Round 2

[0245] The pooled cells are added to the Ligation Master Mix, which is loaded into the Round 2 Plate. An in situ ligation reaction adds a well-specific barcode to the 3 ’ end of the cDNA. The ligation reaction is quenched with Round 2 stop buffer, and the cells are pooled and strained.

[0246] To add round 2 barcodes: 1. On ice, prepare the Round 2 Ligation Master Mix by combining the following with the samples in Resuspension Buffer prepared in Section 1.2: Sample in Resuspension Buffer (2 mL), Round 2 Ligation Buffer (1.95 mL), Round 2 Ligation Enzyme (20 mL), for a total volume of 3.97 mL. 2. Mix thoroughly by pipetting lOx with a Pl 000 set to 1000 pL. Store on ice.

[0247] 3. Remove the Round 2 Plate from the thermocycler, place in a PCR tube rack, and centrifuge for 1 minute at 100 x g at 4°C. 4. While secured in a PCR tube rack on a flat surface, remove the plate seal from the Round 2 Plate and store on ice. 5. Transfer the Round 2 Ligation Master Mix to a basin with a Pl 000.

[0248] 6. With the Round 2 Plate on ice and the basin on the bench, transfer Round 2Ligation Master Mix to each well in the Round 2 Plate as follows: i. Mix the sample in the basin by pipetting 2x with a multichannel P200 set to 40 pL. ii. Transfer 40 pL of the mix to row A of the Round 2 Plate and mix by pipetting 2x. iii. Repeat i-ii to mix the sample in the basin then transfer to rows B-H. Note: If the volume is insufficient to transfer the last row with a multichannel, tilt the basin and transfer the remaining volume with a single channel pipette. If the volume is still insufficient to fill every well, a few can be left empty without impacting experimental results.

[0249] 7 While secured in a PCR tube rack on a flat surface, add a new plate seal to the Round 2 Plate. 8. Place the Round 2 Plate into a thermocycler and run the program shown in Table 21. Upon completion, proceed immediately to the next step.

[0250] 9. Briefly vortex the Round 2 stop buffer and ensure there is no precipitate.Transfer the entire volume of this tube to a new basin with a P1000. 9. Remove the Round 2 Plate from the thermocycler, place in a PCR tube rack, remove the plate seal, and store on ice. 10. With the Round 2 Plate on ice and the basin on the bench, transfer 10 pL of the Round 2 stop buffer to each well in the Round 2 Plate with a multichannel P20. After each transfer, mix by pipetting exactly 3x. 11. While secured in a PCR tube rack on a flat surface, add a new plate seal to the Round 2 Plate. 12. Place the Round 2 Plate into a thermocycler and run the program shown in Table 22. Proceed to the next step while the program is still running.

[0251] 14. Place the Round 3 Plate into a second thermocycler and run the program shown in Table 23. Proceed to the next step while the program is still running.

[0252] 15. Immediately upon completion of the Round 2 Stop program, transfer theRound 2 Plate from the thermocycler to a PCR tube rack, remove the plate seal, and store on ice. 16. With the Round 2 Plate on ice and the basin on the bench, transfer all the liquid in the Round 2 Plate into a new basin as follows: i. With a multichannel P200 set to 50 pL, mix the sample in row A by pipetting 3x. ii. Transfer 50 pL from row A to the basin, iii. Repeat i-ii for rows B-H to mix the sample then transfer to the basin, iv. Transfer any residual liquid in the Round 2 Plate to the basin with a multichannel P20 set to 10 pL.

[0253] 17. Pipette the sample through a cell strainer into a new basin with a Pl 000.Before each transfer, gently mix the cells in the basin by pipetting 2x and tilt the basin to recover as much liquid as possible. 18. Proceed immediately to Section 1.4 (Barcoding Round 3).1.4 Barcoding Round 3

[0254] The Round 3 Ligation Enzyme is added to the pooled cells / nuclei, which are then loaded into the Round 3 Plate. A second in situ ligation reaction adds a third wellspecific barcode, the Illumina Truseq R2 sequence, and a biotin. The sample is then pooled and strained.

[0255] To add round 3 barcodes: 1. Add 20 pL of Round 3 Ligation Enzyme to the basin containing the strained sample. 2. Mix by gently pipetting 20x with a Pl 000 set to 1000 pL. 3. Remove the Round 3 Plate from the thermocycler, place in a PCR tube rack, and centrifuge for 1 minute at 100 x g at 4°C. 4. While secured in a PCR tube rack on a flat surface, remove the plate seal from the Round 3 Plate.

[0256] 5. With the Round 3 Plate on ice and the basin on the bench, transfer 50 pL from the basin to each well in the Round 3 Plate as follows: i. Mix the sample in the basin by pipetting 2x with a multichannel P200 set to 50 pL. ii. Transfer 50 pL of the mix to row A of the Round 3 Plate and mix by pipetting 2x. iii. Repeat i-ii to mix the sample in the basin then transfer to rows B-H. Note: If the volume is insufficient to transfer the last row with a multichannel, tilt the basin and transfer the remaining volume with a single channel pipette. If the volume is still insufficient to fill every well, a few can be left empty without impacting experimental results. 6. While secured in a PCR tube rack on a flat surface, add a new plate seal to Round 3 Plate. 7. Place the Round 3 Plate into a thermocycler and run the program shown in Table 24.

[0257] 8. Briefly vortex the Round 3 stop buffer. Transfer the entire volume to a new basin with a P1000. 9. Remove the Round 3 Plate from the thermocycler, place in a PCR tube rack, remove the plate seal, and store on ice. 10. With the Round 3 Plate on ice and the basin on the bench, transfer 20 pL of the Round 3 stop buffer from the basin to each well in the Round 3 Plate with a multichannel P20. After each transfer, mix by pipetting exactly 3x. 11. Without incubation, proceed immediately to the next step.

[0258] 12. With the Round 3 Plate on ice and the basin on the bench, transfer all the liquid in the Round 3 Plate into a new basin as follows: i. With a multichannel P200 set to 70 pL, mix the sample in row A by pipetting 3x. ii. Transfer 70 pL from row A to the basin, iii. Repeat i-ii for rows B-H to mix the sample then transfer to the basin, iv. Transfer any residual liquid in the Round 3 Plate to the basin with a multichannel P20 pipette set to 10 pL. 13. Pipette the sample through a cell strainer into a new 15 mL tube with a Pl 000. Before each transfer, gently mix the cells in the basin by pipetting 2x and tilt the basin to recover as much liquid as possible. 14. Proceed immediately to section 1.5 (Lysis and Sublibrary Generation).Lysis and Sublibrary Generation

[0259] The cell pool is centrifuged, washed, and resuspended in pre-lysis dilution buffer. The cells are counted and divided into sublibraries. These sublibraries are lysed and stored at -80°C. To generate and lyse sublibraries: 1. Add 70 pL of spin additive to the 15 mL tube with the sample. Gently invert once to mix. 2. Centrifuge the 15 mL tube in a swinging bucket rotor for 5-10 minutes at 200-500 x g at 4°C. Immediately move to the next step after centrifugation. 3. Remove the supernatant until about ~40 pL of liquid remains above the pellet. Use a P1000 for the first 6 mL and then a P200 for the remaining volume. Depending on the number of cells and sample type, the pellet may or may not bevisible. 4. Fully but gently resuspend the pellet in 1 mL Pre-Lyse Wash Buffer. 5. Add an additional 3 mL of Pre-Lyse Wash Buffer for a total addition of 4 mL. 6. Centrifuge the 15 mL tube in a swinging bucket rotor for 5-10 minutes at 200-500 x g at 4°C. Immediately move to the next step after centrifugation. 7. Remove the supernatant until about ~40 pL of liquid remains above the pellet. Use a Pl 000 for the first 3 mL and then a P200 for the remaining volume. 8. Fully but gently resuspend the pellet in 60 pL of Pre-Lysis Dilution Buffer for a final total volume of 100 pL. Store on ice. 9. While minimizing time on ice, count the number of cells in the sample with a hemocytometer or alternative cell counting device. Record the cell count.

[0260] 10. Decide how to divide cells / nuclei across the sublibraries. Do not add more than 12,500 cells / nuclei to a sublibrary. Adding additional cells will result in an increased multiplet rate. 11. Ensure the cells are in suspension by pipetting 5x with a P200 set to 75 pL prior to each transfer. Add the appropriate volume of sample to 8 different 0.2 mL PCR tubes strips. 12. Add the appropriate volume of Pre-Lysis Dilution Buffer to the 0.2 mL tubes for a total volume of 25 pL. 13. Prepare the Lysis Master Mix in a new 1.5 mL tube by adding 220 mL of lysis buffer and 44 ml of lysis enzyme, for a total volume of 264 mL. Mix by pipetting 3x with a P 1000 set to 220 pL. Store at room temperature.

[0261] 14. Add 30 pL of Lysis Master Mix to each 0.2 mL tube with diluted cells.Store at room temperature. 15. Vortex the 0.2 mL tube(s) for 10 seconds. Briefly centrifuge. 16. Place the tube(s) into a thermocycler and run the program shown in Table 25. If continuing to Section 2 without freezing the sample, proceed to Section 2 while the program is still running. 17. Freeze the lysate(s) at -80°C or proceed to Section 2. Safe stopping point: Sublibrary lysates can be stored at -80°C for up to 6 months.2: cDNA Capture and Amplification2.1. cDNA Capture

[0262] The barcoded cDNA is captured with streptavidin-coated magnetic beads and washed to remove cellular debris. To capture the cDNA: 1. Fill an ice bucket. 2. For each lysate, prepare 400 pL of 85% ethanol with nuclease-free water. 3. Equilibrate 80 pL of SPRI beads per lysate to room temperature 4. Remove the desired tube(s) of lysate from the thermocycler (if continuing directly from Section 1) or from storage at -80°C. 5. If previously frozen, incubate the tube(s) in water bath or thermocycler at 37°C for 5 minutes. 6. Briefly centrifuge and store at room temperature. 7. Briefly centrifuge Capture Enhancer and gently mix by pipetting 2x with a P20 set to 15 pL. 8. Add 2.5 pL of Capture Enhancer to each tube of lysate and mix by pipetting 5x with a P200 set to 40 pL. 9. Briefly centrifuge. 10. Incubate for 10 minutes at room temperature. 11. Proceed immediately to the next step during the incubation. Note: This incubation can be extended by 5 additional minutes up to a total of 15 minutes without negatively impacting performance.

[0263] 12. Vortex Streptavidin Beads until fully mixed. Add the appropriate volume of Streptavidin Beads to a new 1.5 mL tube depending on the number of lysates being processed, e.g., 1 lysate being processed: 44 mL of streptavidin beads; 8 lysates being processed: 352 mL of Streptavidin Beads. 13. Place the tube on the magnetic rack for 1.5 mL tubes until the solution clears (~2 minutes). 14. Remove and discard the supernatant. 15. Remove the tube from the magnetic rack and fully resuspend the bead pellet in the appropriate volume of Bead Wash Buffer: 1 lysate being processed: 50 mL bead wash buffer; 8 lysates being processed: 400 mL bead wash buffer.

[0264] 16. Place the tube on the magnetic rack for 1.5 mL tubes until the solution clears (~2 minutes). 17. Remove and discard the supernatant. 18. Repeat steps 15-17 twice for a total of 3 washes. 19. Remove the tube from the magnetic rack. Fully resuspend the pellet in the appropriate volume of binding buffer as follows and store at room temperature: 1 lysate being processed: 55 mL of binding buffer; 8 lysates being processed: 440 mL of binding buffer.

[0265] 20. Add 50 pL of Streptavidin Beads in Binding Buffer to each tube of lysate and fully mix by pipetting 2x with a P200 set to 90 pL. 21. Place the tube(s) into a 96 well PCR tube rack, press to secure, and ensure the caps are secured tightly. Place the lid on therack. 22. Place the rack onto a vortex mixer with a plate adapter. Push to secure. Vortex on 20% power (-800-1000 RPM) for 30 minutes at room temperature. Note: To ensure the beads are being mixed sufficiently, check that the beads are in solution 10 minutes into the incubation. If settled, increase the vortex mixing speed to keep the beads in solution. 23. Remove the tube(s) from the vortex mixer. 24. Briefly vortex the tube(s) on a standard vortex adapter. Briefly centrifuge without letting beads collect at the bottom of the tube(s). 25. Place the tube(s) on the high magnet position of the Parse Biosciences Magnetic Rack, so the magnet is closer to the top of the 0.2 mL tubes. Incubate until the solution clears (-2 minutes).

[0266] 26. While still on the magnetic rack, remove and discard the supernatant. 27.Remove the tube(s) from the magnetic rack. Fully resuspend each bead pellet with 125 pL Wash Buffer 1. 28. Incubate for 1 minute at room temperature. 29. Return the tube(s) to the high position of the magnetic rack. Incubate until the solution clears (-2 minutes). 30. While still on the magnetic rack, remove and discard the supernatant. 31. Repeat steps 27- 30 once for a total of 2 washes with Wash Buffer 1. 32. Remove the tube(s) from the magnetic rack. Fully resuspend each bead pellet with 125 pL Wash Buffer 2. 33. Incubate for 1 minute at room temperature. 34. Proceed immediately to Section 2.2 (cDNA template switch).2.2 cDNA Template Switch

[0267] After an additional wash, the template switch master mix is added to the captured cDNA. The template switch reaction adds a 5’ adaptor to the cDNA. To perform template switch: 1. Prepare the Template Switch Master Mix in a new 1.5 mL tube as shown in Table 26, depending on the number of sublibraries being processed. 2. Mix by pipetting lOx and store on ice.

[0268] 3. Place each tube of captured cDNA from Section 2.1 on the high position of the magnetic rack. Incubate until the solution clears (~2 minutes). 4. While still on the magnetic rack, remove and discard the supernatant. 5. While still on the magnetic rack, add 125 pL of Wash Buffer 3 to each tube. 6. Incubate for 1 minute at room temperature. 7. While still on the magnetic rack, remove and discard the Wash Buffer 3. 8. Remove the tube(s) from the magnetic rack. Fully resuspend each bead pellet with 100 pL of the Template Switch Master Mix. 9. Briefly centrifuge without letting beads collect at the bottom of the tube(s). 10. Incubate for 30 minutes at room temperature. 11. Fully resuspend each bead pellet by mixing 5x with a P200 set to 75 pL. 12. Place the tube(s) into a thermocycler and run the program shown in Table 27.

[0269] 13. Proceed immediately to Section 2.3. Alternatively, proceed to step 14 to store samples prior to cDNA amplification. 14. Place the tube(s) on the high position of the magnetic rack. Incubate until the solution clears (~2 minutes). 15. While still on the magnetic rack, remove and discard the supernatant. 16. Remove the tube(s) from the magnetic rack. Fully resuspend each bead pellet with 125 pL wash buffer 2. Safe stopping point: Template switched cDNA can be stored at 4°C for up to 18 hours. Do not freeze.2.3 cDNA Amplification

[0270] The captured cDNA is washed and amplified with Template Switch Primerand Illumina Truseq R2- specific primers. To amplify the cDNA: 1. Prepare the Amplification Reaction Solution Master Mix in a new 1.5 mL tube as shown in Table 28. 2. Mix by pipetting lOx and store on ice.

[0271] 3. Place each tube of template switched cDNA from Section 2.2 on the high position of the Parse Biosciences Magnetic Rack. Incubate until the solution clears (~2 minutes). 4. While still on the magnetic rack, remove and discard the supernatant. 5. While still on the magnetic rack, add 125 pL of wash buffer 3 to each tube. 6. Incubate for 1 minute at room temperature. 7. While still on the magnetic rack, remove and discard the wash buffer 3. 8. Remove tube(s) from the magnetic rack. Fully resuspend each bead pellet with 100 pL of the Amplification Master Mix. Store on ice. 9. Determine the number of PCR cycles required for cDNA amplification based on the recommendations in Table 29. Although these recommendations are appropriate for many cell types, the number of cycles may need to be optimized for your sample type.

[0272] 10. Place the tube(s) into a thermocycler and run the program shown in Table30. If processing sublibraries with different numbers of cells / nuclei, they should be amplified in separate thermocyclers according to the recommendations in Table 29. Safe stopping point: Amplified cDNA can be stored at 4°C for up to 18 hours.2.4 Post-Amplification Purification

[0273] Amplified cDNA is purified with a 0.8x SPRI bead cleanup. To purify the cDNA: 1. Gather 400 pL of freshly prepared 85% ethanol per tube of amplified cDNA with nuclease-free water. 2. Gather room temperature SPRI beads (80 pL per tube of amplified cDNA). 3. Place each tube of amplified cDNA on the high position of the magnetic rack. Incubate until the solution clears (~2 minutes). 4. While still on the magnetic rack, transfer 90 pL of the supernatant containing the cDNA into a new 0.2 mL tube(s). Store at room temperature. 5. Vortex the SPRI beads until fully mixed. Add 72 pL of SPRI beads to each tube with amplified cDNA. 6. Vortex the tube(s) for 5 seconds. Briefly centrifuge. 7. Incubate for 5 minutes at room temperature. 8. Place the tube(s) on the high position of the magnetic rack. Incubate until the solution clears (~2 minutes). 9. While still on the magnetic rack, remove and discard the supernatant.

[0274] 10. While still on the magnetic rack, add 180 pL of 85% ethanol to each tube.11. Incubate for 1 minute at room temperature. 12. While still on the magnetic rack, remove and discard the supernatant. 13. Repeat steps 10-12 once for a total of 2 washes. Remove any residual ethanol with a P20. 14. While still on the magnetic rack, air dry the SPRI beads (~2 minutes). 15. Remove the tube(s) from the magnetic rack. Fully resuspendeach bead pellet with 25 pL of nuclease-free water. 16. Incubate for 10 minutes at 37°C in a thermocycler. 17. Place the tube(s) on the low magnet position of the magnetic rack, so the magnet is closer to the bottom of the 0.2 mL tubes. Incubate until the solution clears (~2 minutes). 18. While still on the magnetic rack, transfer 25 pL of the supernatant containing the purified cDNA into new 0.2 mL tube(s). Store on ice. Safe stopping point: Amplified cDNA can be stored at 4°C for up to 48 hours or at -20°C for up to 3 months.2.5 cDNA Quantification

[0275] The concentration and size distribution of the cDNA are measured with fluorescent dyes and capillary electrophoresis. The cDNA is then stored at 4°C for up to 48 hours or at -20°C for up to 3 months. To quantify the cDNA: 1. Measure the concentration of each tube of purified cDNA from section 2.4 with ., the Qubit dsDNA HS (High Sensitivity) Assay Kit according to the manufacturer’s instructions. Record the concentration(s). 2. Assess the size distribution of each tube of purified cDNA with a High Sensitivity DNA Kit on the Agilent Bioanalyzer System or High Sensitivity D5000 ScreenTape and Reagents on the Agilent TapeStation System according to the manufacturer’s instructions. Safe stopping point: purified cDNA can be stored at 4°C for up to 48 hours or at -20°C for up to 3 months. Otherwise, proceed immediately to Section 3 (Sequencing Library Preparation). Exemplary post-amplification sublibrary cDNA size distributions are shown in FIGS. 10A-10B. Note: Samples may need to be diluted to be within the manufacturer's recommended concentration range. Typically, between a 1:3 to 1: 10 dilution is appropriate. Note: The traces shown in FIGS. 10A-10B are representative of typical TapeStation cDNA traces. However, the shape and prominence of the trace is dependent on cell type, sublibrary size, and amount of DNA loaded into the TapeStation. Accordingly, sublibraries with minor deviations can still produce high quality data and are suitable for use in the present methods.3. Sequencing Library Preparation3.1. Fragmentation and End Prep

[0276] Barcoded and amplified cDNA is fragmented, end repaired and A-tailed in a single reaction. To prepare for sequencing library preparation: 1. Prepare 1.2 mL 85% ethanol per sublibrary with nuclease-free water. 2. Equilibrate 180 pL of SPRI beads per sublibrary to room temperature. 3. Fill an ice bucket. 4. Take out the magnetic rack for 0.2mL PCR tubes. 5. Obtain recorded cDNA concentrations from section 2.5. 6. Vortex the tube(s) of cDNA for 5 seconds. 7. Briefly centrifuge. 8. Prepare diluted cDNA in new 0.2 mL tube(s) as follows to a final volume of 35 pL and store on ice: 10 mL purified cDNA, 25 mL nuclease-free water, for total volume of 35 pL. Store any remaining sublibrary cDNA at -20°C. Start the program shown in Table 31.

[0277] 10. Vortex the Fragmentation Buffer for 5 seconds. Briefly centrifuge. 11.Prepare the Fragmentation Master Mix in a new 1.5 mL tube as shown in Table 32. Mix by pipetting lOx and store on ice.

[0278] 12. Add 15 pL of Fragmentation Master Mix to each tube of diluted cDNA.Mix by pipetting lOx with a P200 multichannel pipette set to 40 pL. Briefly centrifuge. 13. Place the tube(s) into a cooled thermocycler (held at step 1 of the program shown in Table 31) and advance to step 2 of the program. 14. As soon as the program reaches step 4 of the thermal cycling program(4 °C), store the tube(s) on ice and proceed immediately to section 3.2 (Post-Fragmentation and End Prep Size Selection).3.2. Post-Fragmentation and End Prep Size Selection

[0279] The fragmented and end prepped DNA is size selected with a double-sided SPRI cleanup. To size select the fragmented and end prepped DNA: 1. Gather freshly prepared 85% ethanol. 2. Gather room temperature SPRI beads (~50 pL per sublibrary). 3. Vortex the SPRI beads until fully mixed. Add 30 pL of SPRI beads to each tube of fragmented and end prepped DNA. 4. Vortex the tube(s) for 5 seconds. Briefly centrifuge. 5. Incubate for 5 minutes at room temperature. 6. Place the tube(s) on the high position of the magnetic rack for 0.2 mL tubes. Incubate until the solution clears (~2 minutes). 7. While still on the magnetic rack, transfer 75 pL of the supernatant containing the fragmented and end prepped DNA into new 0.2 mL tube(s). Discard the tube(s) with bead pellet(s). 8. Add 10 pL of SPRI beads to each tube. 9. Vortex the tube(s) for 5 seconds. Briefly centrifuge. 10. Incubate for 5 minutes at room temperature.

[0280] 11. Place the tube(s) on the high position of the magnetic rack for 0.2 mL tubes.Incubate until the solution clears (~3 minutes). 12. While still on the magnetic rack, remove and discard the supernatant. 13. While still on the magnetic rack, add 180 pL of 85% ethanol to each tube. 14. Incubate for 1 minute at room temperature. 15. While still on the magnetic rack, remove and discard the supernatant. 16. Repeat steps 13-15 once for a total of 2 washes. Remove any residual ethanol with a P20. 17. While still on the magnetic rack, air dry the SPRI beads (~30 seconds). 18. Remove the tube(s) from the magnetic rack. Fully resuspend each bead pellet with 50 pL of nuclease-free water. 19. Incubate for 5 minutes at room temperature. 20. Place the tube(s) on the high position of the magnetic rack. Incubate until the solution clears (~2 minutes). 21. While still on the magnetic rack, transfer 50 pL of the supernatant into new 0.2 mL tube(s). Safe stopping point: The size-selected fragmented and end prepped DNA can be stored at 4°C for up to 18 hours or at -20°C for up to 2 weeks.3.3. Adaptor Ligation

[0281] Adaptors with an Illumina Truseq R2 sequence are ligated to the 5’ end of the fragmented and end prepped DNA. To ligate adaptors: 1. Prepare the Adaptor Ligation Master Mix in a new 1.5 mL tube as shown in Table 33. 2. Mix by pipetting lOx and store on ice.

[0282] 3. Add 50 pL of Adaptor Ligation Master Mix to each tube of purified fragmented and end prepped DNA from section 3.2. Mix by pipetting lOx with a P200 multichannel pipette set to 80 pL. Briefly centrifuge. 4. Place the tube(s) into a thermocycler and run the program shown in Table 34. 5. As soon as the program reaches 4°C, store the adapter-ligated DNA on ice and proceed immediately to section 3.4 (Postligation purification).3.4. Post-Ligation Purification

[0283] Adaptor ligated DNA is purified with a 0.8x SPRI bead cleanup. To purify the ligated DNA: 1. Gather freshly prepared 85% ethanol. 2. Gather room temperature SPRI beads (~90 pL per sublibrary). 3. Vortex the SPRI beads until fully mixed. Add 80 pL of SPRI beads to each tube of adaptor ligated DNA from section 3.3. 4. Vortex the tube(s) for 5 seconds. Briefly centrifuge. 5. Incubate for 5 minutes at room temperature. 6. Place the tube(s) on the high position of the magnetic rack for 0.2 mL tubes. Incubate until the solution clears (~2 minutes). 7. While still on the magnetic rack, remove and discard the supernatant. 8. While still on the magnetic rack, add 180 pL of 85% ethanol to each tube. 9. Incubate for 1 minute at room temperature. 10. While still on the magnetic rack, remove and discard the supernatant.

[0284] 11. Repeat steps 8-10 once for a total of 2 washes. Remove any residual ethanol with a P20. 12. While still on the magnetic rack, air dry the SPRI beads (~2 minutes). 13. Remove the tube(s) from the magnetic rack. Fully resuspend each bead pellet with 23 pL of nuclease-free water. 14. Incubate for 5 minutes at room temperature. 15. Place the tube(s) on the low magnet position of the magnetic rack so the magnet is closer to the bottom of the 0.2 mL tubes. Incubate until the solution clears (~2 minutes). 16. While still on the magnetic rack, transfer exactly 21 pL of the supernatant containing the purified adaptor ligated DNA into new 0.2 mL tube(s). Store on ice. 17. Proceed immediately to section 3.5 (Barcoding Round 4).3.5. Barcoding Round 4

[0285] Purified adaptor ligated DNA is PCR amplified with Illumina TruSeq R1 and R2 primers. This indexing PCR generates sequencing libraries and adds i5 / i7 UDIs that act as a fourth cell barcode. To add round 4 barcodes: 1. Centrifuge the UDI Plate - WT at 100 x g for 1 minute. 2. Wipe the surface of the plate with 70% ethanol and allow to dry. 3. Orient the UDI Plate - WT with the notch on the bottom left. For each sublibrary being processed, choose one unused well of the UDI Plate - WT and record the well position and number for each sublibrary. 4. With a multichannel P20, pierce the seal of the chosen wells in the UDI Plate - WT. 5. With a multichannel P20 and new tips, mix by pipetting 5x then immediately transfer 4 pL from a chosen unused well of the UDI Plate - WT to its corresponding tube of purified adaptor ligated DNA from section 3.4. 6. If any unused wells remain in the UDI Plate - WT, store the plate at -20°C. Do not reuse wells. 7. Add 25 pL of Library Amp Mix to each tube. Mix by pipetting lOx with a P200 multichannel pipette set to 25 pL. Briefly centrifuge. 8. Determine the number of PCR cycles required for indexing PCR based on the amount of cDNA added to the fragmentation and end prep reaction as recorded in section 2.5, according to the recommendations shown in Table 35. The UDI sequences in the UDI Plate - WT are shown in Table 35.

[0286] Place the tube(s) into a thermocycler and run the program shown in Table 36. If processing sublibraries with different cDNA concentrations, they should be amplified in separate thermocyclers according to the recommendations in Table 36. Safe stopping point: Prior to size selection, sequencing libraries can be stored at 4°C for up to 18 hours.3.6. Post-Round 4 Barcoding Size Selection

[0287] The sequencing libraries are size selected with a double sided SPRI cleanup. To size select the sequencing libraries: 1. Gather freshly prepared 85% ethanol. 2. Gather room temperature SPRI beads (~50 pL per sublibrary). 3. Vortex the SPRI beads until fully mixed. Add 30 pL of SPRI beads to each sequencing library tube. 4. Vortex the tube(s) for 5 seconds. Briefly centrifuge. 5. Incubate for 5 minutes at room temperature. 6. Place the tube(s) on the high position of the magnetic rack for 0.2 mL tubes. Incubate until the solution clears (~2 minutes). 7. While still on the magnetic rack, transfer 75 pL of the supernatant containing theDNA into new 0.2 mL tube(s). Discard the tube(s) with bead pellet(s). 8. Add 10 pL of SPRI beads to each tube. 9. Vortex the tube(s) for 5 seconds. Briefly centrifuge. 10. Incubate for 5 minutes at room temperature. 11. Place the tube(s) on the high position of the magnetic rack for 0.2 mL tubes. Incubate until the solution clears (~3 minutes). 12. While still on the magnetic rack, remove and discard the supernatant. 13. While still on the magnetic rack, add 180 pL of 85% ethanol to each tube. 14. Incubate for 1 minute at room temperature. 15. While still on the magnetic rack, remove and discard the supernatant. 16. Repeat steps 13-15 once for a total of 2 washes. Remove any residual ethanol with a P20. 17. While still on the magnetic rack, air dry the SPRI beads (~30 seconds). 18. Remove the tube(s) from the magnetic rack. Fully resuspend each bead pellet with 20 pL of nuclease-free water. 19. Incubate for 5 minutes at room temperature. 20. Place the tube(s) on the low position of the magnetic rack. Incubate until the solution clears (~2 minutes). 21. While still on the magnetic rack, transfer the supernatant into new 0.2 mL tube(s). Store on ice. Safe stopping point: Sequencing libraries can be stored at -20°C for up to 3 months.3. 7. Sequencing Library Quantification

[0288] The concentration and size distribution of the sequencing libraries are measured with fluorescent dyes and capillary electrophoresis. To quantify the sequencing libraries: 1. Measure the concentration of each purified sequencing library from section 3.6 with, e.g., a Qubit dsDNA HS (High Sensitivity) Assay Kit according to the manufacturer’s instructions. 2. Assess the size distribution of each purified sequencing library with a High Sensitivity DNA Kit on the Agilent Bioanalyzer System or High Sensitivity DI 000 ScreenTape and Reagents on the Agilent TapeStation System according to the manufacturer’ s instructions. Note: Samples may need to be diluted to be within the manufacturer's recommended concentration range. Typically, between a 1 :3 to 1 : 10 dilution is appropriate. Safe stopping point: Sequencing libraries can be stored at -20°C for up to 3 months. FIGS. 11A-11B provide exemplary size distributions of the sequencing libraries prior to Illumina Sequencing.

[0289] Note: The traces shown in FIGS. 11A-11B are representative of typical TapeStation of DNA from indexed sublibraries. There should be a peak between 400-500 bp. The prominence of the trace is dependent on the amount of DNA loaded into the TapeStation. Sublibraries with minor deviations can still produce high quality data. In addition, if using a Bioanalyzer, there may be an additional peak present. This typically occurs if products areoveramplified, but it should not impact sequencing or data quality (assuming there is still a peak present at 400-500 bp). Do not use this additional peak when estimating amplicon size.Section 4: BCR Library Preparation4.1. Pool Libraries

[0290] Libraries are pooled and prepared for the hybridization reaction. Up to 8 indexed sublibraries can be used for each BCR reaction (e.g. with a BCR WT kit; in other embodiments, e.g., when using BCR mini or BCR mega kits, fewer or more sublibraries may be used). No more than 12 pL per sublibrary should be used to prepare the BCR library to ensure that there is sufficient input for WT sequencing. When pooling sublibraries in step 10, vacuum concentration can be used rather than SPRI bead concentration.

[0291] To prepare for BCR library preparation: 1. Fill a bucket with ice. 2. Set a heat block to 65°C. 3. Program a thermocycler to 95°C and set the heated lid to 105°C. 4. Equilibrate SPRI beads (Ampure XP or KAPA Pure Beads) to room temperature for at least 30 minutes. 5. Prepare at least 700 pL of 85% ethanol per reaction and the magnetic rack for 1.5 mL tubes.

[0292] 6. Incubate the Hybridization Mix in the heat block at 65°C for 10 minutes or until no precipitate is present before proceeding. 7. Incubate the Hybridization Mix at room temperature for 5 minutes to equilibrate the solution. 8. Vortex sublibraries (2-3 seconds). 9. Briefly centrifuge (~2 seconds). 10. Pool up to 8 indexed sublibraries (when using, e.g., a BCR WT kit) into a 1.5 mL tube to make a Library Pool. We recommend adding 100 ng of each sublibrary going into BCR library preparation for a maximum of 800 ng of input per tube. 11. If the total volume of the Library Pool is less than 100 pL, bring the volume up to 100 pL with nuclease-free water. 12. Vortex the SPRI beads until fully mixed. Add 1.8x of SPRI beads to the Library Pool. Note: For example, if a Library Pool is 100 pL, add (1.8 x 100 pL) = 180 pL of SPRI beads. 13. Vortex the tube for 5 seconds. Briefly centrifuge (~2 seconds). 14. Incubate tube at room temperature for 5 minutes.

[0293] 15. Place the tube in a 1.5 mL magnetic rack and wait for all the beads to bind to the magnet (~3 minutes: ensure the liquid is clear). 16. With the tube still on the magnetic rack, slowly remove and discard the clear supernatant. 17. Without resuspending beads, add 300 pL of 85% ethanol to the tube using a P1000 and wait 1 minute. 18. Using a pipette, aspirate and discard the ethanol from the tube. 19. Without resuspending beads, add another 300 pL of 85% ethanol to the tube using a Pl 000 and wait 1 minute. 20. Using a pipette, aspirate and discardall of the ethanol from the tube (it may be necessary to remove the final few drops with a P20 pipette). With the tube still on the rack, air dry the beads (~2 minutes). 21. Remove tube from the magnetic rack. Proceed directly to Section 4.2.4.2. Blocking & Hybridization

[0294] Blockers are added to the library pool to reduce off-target binding. Next, during hybridization, biotinylated probes bind to the BCR repertoire. 1. Add the following reagents individually to the Library Pool from step 4.1.21 : Evercode Blocker Solution (7 mL), Blocker Solution (5 mL), Total volume: 12 mL. Resuspend the beads by gentle pipetting with a P20 set to 10 mL. 3. Briefly centrifuge and carefully transfer the entire volume (12 pL) into a new 0.2 mL tube using a P20 set to 15 pL. 4. Heat the Library Pool for 5 minutes at 95°C in a thermocycler with the lid heated to 105°C, then immediately place at room temperature. 5. In a new 0.2 mL tube, prepare a Probe Solution as follows: Hybridization Mix (21 mL), Human or Mouse BCR Panel (4.2 mL), Nuclease-Free Water (4.2 mL), total volume (29.4 mL). Mix by pipetting.

[0295] 6. Heat the Probe Solution for 2 minutes at 95°C in a thermocycler with the lid heated to 105°C, then immediately cool for 5 minutes on ice. 7. Separately, incubate both the Probe Solution and Library Pool at room temperature for 5 minutes. 8. Set a thermocycler to 70°C with the lid at 85°C for infinite time. 9. Vortex the Probe Solution for 5 seconds. Briefly centrifuge (~2 seconds). 10. Transfer the entire volume (28 pL) of the Probe Solution into the Library Pool to create the Hybridization Reaction. 11. Vortex the tube for 5 seconds. Briefly centrifuge (~2 seconds). 12. Without mixing, add 30 pL of Hybridization Enhancer to the top of the meniscus of the Hybridization Reaction from step 11. 13. Briefly centrifuge tube to ensure there are no bubbles present. 14. Ensure tubes are tightly capped to prevent excess evaporation. 15. Incubate the Hybridization Reaction (72 pL) at 70°C for 15-17 hours in a thermocycler with the lid at 85°C. Note: Hybridizing for less than 15 hours or more than 17 hours may impact hybridization efficiency.5. BCR Amplification5.1 Bind and Wash

[0296] Streptavidin binder beads capture the hybridized BCR repertoire prior to amplification. To prepare BCR capture pool for amplification: 1. Gather two heat blocks, set one to 68°C (for 1.5 mL tube) and the other to 48°C (for 2 mL and 5 mL tubes). 2. EquilibrateStreptavidin Binder Beads and the SPRI beads (Ampure XP or KAPA Pure Beads) to room temperature for at least 30 minutes. 3. Prepare at least 400 pL of 85% ethanol. 4. Prepare the magnetic rack for 1.5 mL tubes.

[0297] 6. Check if precipitate is dissolved, then equilibrate the Streptavidin BindingBuffer to room temperature. Keep the Bead Wash Buffer A and Bead Wash Buffer B in their respective heat blocks. 7. Ensure the Streptavidin Binder Beads have been equilibrated to room temperature for at least 30 minutes. Vortex the Streptavidin Binder Beads until mixed. 8. Add 100 pL of Streptavidin Binder Beads to a new 1.5 mL tube for the hybridization reaction. 9. Add 200 pL of Streptavidin Binding Buffer to the tube with Streptavidin Binder Beads. Mix thoroughly by pipetting lOx with a P200 set to 200 pL. 10. Place the tube in a 1.5 mL magnetic rack and wait for all the beads to bind to the magnet (~2 minutes: liquid should be clear). 11. Using a pipette, aspirate and discard the supernatant from the tube. Remove tube from the magnetic rack. 12. Repeat steps 9-11 twice for a total of three washes.

[0298] 13. Add 200 pL of Streptavidin Binding Buffer and resuspend the beads by vortexing until fully homogenized. 14. Heat the resuspended beads at 68°C for 10 minutes before proceeding to the next step. 15. Set a P200 pipette to 80 pL. After the hybridization reaction from Section 4.2 is complete, open the thermocycler lid and immediately transfer the entire volume of the Hybridization Reaction into a corresponding tube of preheated Streptavidin Binder Beads from step 5.1.14. Mix by pipetting. Note: The rapid transfer from the 70°C thermocycler in step 4.2.15 is a critical step for minimizing off-target binding. Do not remove the Hybridization Reaction tube from the thermocycler or otherwise allow it to cool to less than 70°C before transferring the solution to the washed Streptavidin Binding Beads. Allowing the Hybridization Reaction to cool to room temperature for more than 5 minutes may result in as much as 10-20% increase in off-target binding. 16. Incubate the tube containing the Hybridization Reaction and Streptavidin Binder Beads mixture at 68°C for 5 minutes. 17. Remove the tube from the heat block. Do not vortex, briefly centrifuge (~2 seconds).

[0299] 18. Place the tube in a magnetic rack and wait for all the beads to bind to the magnet(~2 minutes: liquid should be clear). 19. Using a pipette, aspirate and discard the clear supernatant from the tube. Note: Some Hybridization Enhancer reagent residue may be visible after supernatant removal and throughout each wash step. This will not affect the final capture product. 20. Remove the tube from the magnetic rack, add 200 pL of 68°C Bead Wash Buffer A. Mix by pipetting. 21. Incubate the tube at 68°C for 5 minutes and place Bead Wash BufferA back into the 68°C heat block. Briefly centrifuge. 22. For the hybridization reaction, transfer the entire volume from step 21 (-200 pL) into a new 1.5 mL tube. This step reduces background from non-specific binding to the surface of the tube. 23. Place the tube in a 1.5 mL magnetic rack and wait for all the beads to bind to the magnet (-1 minute: liquid should be clear). 24. Using a pipette, aspirate and discard the clear supernatant from the tube. 25. Remove the tube from the magnetic rack and add 200 pL of 48°C Bead Wash Buffer B. Mix by pipetting and briefly centrifuge. 26. Incubate the tube at 48°C for 5 minutes and place Bead Wash Buffer B back into the 48°C heat block. 27. Place the tube in a magnetic rack and wait for all the beads to bind to the magnet (-1 minute: liquid should be clear). 28. Using a pipette, aspirate and discard the clear supernatant from the tube. 29. Repeat steps 25-28 twice for a total of three washes. 30. Remove any residual supernatant with a P20 pipette. Proceed immediately to the next step. 31. Remove the tube from the magnetic rack and resuspend in 45 pL of nuclease- free water. Mix by pipetting until homogenized, then incubate on ice. This solution will be referred to as Streptavidin Binding Bead Slurry. 32. Proceed immediately to Section 5.2.5.2. BCR Amplification

[0300] The BCR repertoire is amplified for sequencing: 1. Prepare 400 pL of 85% ethanol per reaction. 2. Prepare the magnetic rack for 1.5 mL tubes. 3. Mix the Streptavidin Binding Bead Slurry by pipetting. 4. For the hybridization reaction, prepare a PCR mixture in a new 0.2 mL PCR strip as follows: Streptavidin Binding Bead Slurry (22.5 mL), Enrichment Primer Mix (2.5 mL), Enrichment Amplification mix (25 mL), total volume (50 mL). Then, mix by pipetting, centrifuge briefly, and store on ice. 5. Determine the number of PCR cycles required based on Table 38. 6. Place the tube into a thermocycler and run the program shown in Table 39.

[0301] 7. As soon as the program reaches 4°C, place the tube at room temperature and proceed immediately to the next step. 8. Ensure the SPRI beads have been equilibrated to room temperature for at least 30 minutes. Vortex the SPRI beads until fully mixed. Add 90 pL of SPRI beads (1.8x) to the tube. Vortex for 5 seconds. 9. Incubate tube at room temperature for 5 minutes. 10. Place the tube on the high position of the magnetic rack for 0.2 mL tubes and wait for all the beads to bind to the magnet (~2 minutes: liquid should be clear). 11. With tubes still on the magnetic rack, remove and discard the clear supernatant. 12. With tubes still on the magnetic rack, add 180 pL of 85% ethanol to the tube. 13. Incubate tube at room temperature for 1 minute.

[0302] 14. With tubes still on the magnetic rack, remove and discard the supernatant. 15.Repeat steps 12-14 once more for a total of 2 washes. Remove any residual ethanol with a P20 pipette. 16. With tubes still on the magnetic rack, air dry the beads (~30 seconds). 17. Remove the tube from the magnetic rack. Fully resuspend the bead pellet with 32 pL of nuclease-free water. 18. Incubate the tube at room temperature for 5 minutes. 19. Place the tube on the low magnet position of the magnetic rack for 0.2 mL tubes and wait for all the beads to bind to the magnet (~2 minutes: liquid should be clear). 20. With the tube still on the magnetic rack, transfer 30 pL of the supernatant containing the purified BCR sublibraries into a new 0.2 mL tube. Store on ice. 21. Proceed to Section 5.3. Note: sequencing libraries can be stored at -20°C for up to 3 months.5.3 BCR Library Quantification

[0303] The concentration and size distribution of the sequencing libraries are measured with fluorescent dyes and capillary electrophoresis. 1. Measure the concentration of the BCR library from Section 5.2 with, e.g., the Qubit dsDNA HS (High Sensitivity) Assay Kit according to the manufacturer’ s instructions. 2. Assess the size distribution of the purified DNA with, e.g., a High Sensitivity DNA Kit on the Agilent Bioanalyzer System according to the manufacturer’s instructions. FIGS. 12A-12B provide an exemplary size distribution before Illumina Sequencing.

[0304] Note: The traces shown in FIGS. 12A-12B are representative of typical TapeStation of DNA from BCR sublibraries. There should be a peak between 400-500 bp. The prominence of the trace is dependent on the amount of DNA loaded into the Bioanalyzer or TapeStation. Samples with minor deviations can still produce high quality data. In addition, if quantifying DNA with a Bioanalyzer, there may be an additional peak present. If there is a 400- 500 bp peak, it should not impact sequencing or data quality, but care should be taken when calculating the library concentration before sequencingSequencing information

[0305] A minimum sequencing depth of 20,000 reads per cell is recommended for whole transcriptome libraries, and 5,000 reads per cell for the BCR library. However, ideal sequencing depth is dependent on the sample type and experimental goals. For example, if trying to capture rare cell types in a heterogenous population, deeper sequencing is required. Alternatively, if analyzing a homogeneous population, more shallow sequencing may be appropriate. Whole transcriptome sequencing libraries should be diluted and denatured according to the manufacturer’s instructions for the relevant sequencing instrument. We recommend adding 5% PhiX for optimal sequencing quality.

[0306] The diagram shown in FIG. 7 illustrates the composition of the sequencing libraries generated. This library structure applies to both the whole transcriptome library and the BCR-enriched library.

[0307] Libraries should be sequenced with paired reads using the read structure shown in Tables 40-41. Read lengths longer than those recommended below are acceptable, but additional bases in read 2 are trimmed by the Parse Analysis Pipeline. Note that WT andBCR libraries have different sequencing requirements. The WT and BCR libraries should not be run together; however, they can be run in different lanes.

[0308] The fourth barcode that tags each sublibrary act as standard Illumina i7 and i5 indexes. Refer, e.g., to Table 37 to demultiplex the UDI WT and BCR libraries.Example 2, Exemplary protocol for fixation of mouse cells for BCR profilingWorkflow

[0309] From a single cell suspension, the Evercode Mouse BCR Cell Fixation kit generates fixed and permeabilized cells ready for use in Evercode BCR kits. This 1.5 mL tube based workflow is recommended when processing <12 samples at a time. The protocol can be adapted, e.g., for different numbers of samples. For example, if processing >12 samples at a time, a mid-throughput plate-based workflow can be used which streamlines fixation when processing more samples, and if processing >48 samples, a High-Throughput Plate Workflow can be used. The protocol can also be adapted for use with other mammalian cells, including human cells. Alternatively, for human cells a standard Evercode Cell Fixation kit may be used.

[0310] Fixation maintains cell structure, prevents RNA degradation, and locks the RNA inside the cells, which are crucial for downstream processing with Evercode split-pool combinatorial barcoding technology (FIG. 13).

[0311] Because fixed samples are also stable for up to 6 months at -80°C, Evercode Mouse BCR Cell Fixation provides flexibility by separating sample collection from library preparation. It also enables samples to be stored and batched after fixation so they can be processed through library preparation together, reducing the potential of batch effects.

[0312] FIG. 14 provides an overview of the fixation workflow. Between 100,000 and 1 million cells can be fixed in a single reaction. Note that more than 100,000 cells may need to be fixed to fully utilize the capacity of the downstream Evercode kits. If desired, 1-4 million cells can be fixed in a single reaction. Details of the total and hands-on time required for the cell fixation workflow is shown in Table 42.Sample Input

[0313] This protocol begins with a previously prepared single cell suspension. We recommend suspensions with >70% viability (ideally above 90%) and <5% aggregation / debris. If cells were previously frozen, ensure the suspension is completely thawed and in suspension before beginning fixation. We recommend minimizing the length of time samples are stored on ice prior to fixation, as it can negatively impact results. Between 100,000 and 1 million cells can be fixed in a single reaction. However, we recommend using the highest number available up to 1 million total. Exceeding 1 million cells in a single fixation will result in substantially elevated doublet rates. The minimum input into fixation should also be determined based on how the samples will be processed downstream. Table 43 provides guidance on the post-fixation concentrations needed for downstream kits. However, more or less sample input may be required depending on the exact experimental design.

[0314] Note that retention during fixation varies typically between 40-60%, and some cells will be lost when freezing and thawing fixed samples, typically between 5-15%. The final concentration of cells post-fixation is also influenced by the resuspension volume used in Step 18 of Section 2. These factors should all be taken into account when determining how much sample input is needed for fixation.Avoiding RNase Contamination

[0315] Standard precautions should be taken to avoid introducing RNases into samples or reagents throughout the workflow. Always wear proper laboratory gloves and use aseptic technique. Although RNases are not inactivated by ethanol or isopropanol, they are inactivated by products such as RNaseZap RNase Decontamination Solution (Thermo Fisher Scientific). These can be sprayed on benchtops and pipettes. Nuclease-free, filtered pipette tips should be used to reduce RNase contamination from pipettes.Cell Detachment

[0316] If using adherent cell line samples, we recommend TrypLE Express Enzyme (IX), phenol red (Thermo Fisher Scientific). Due to high RNase activity, we do not recommend dissociation with standard trypsin, which may reduce gene and transcript detection.Cell Counting and Quality Assessment

[0317] We recommend a hemocytometer for counting, but alternative counting devices can also be used. If possible, validate counts from alternative devices to a hemocytometer when first using an Evercode Fixation workflow. When first using an Evercode Fixation workflow, we suggest saving images at each counting step. To assess sample quality, we recommendusing viability stains like trypan blue or acridine orange and propidium iodide (AO / PI). After fixation, the cells are permeabilized and should appear dead with viability stains. If using Acridine Orange / Propidium Iodide (AO / PI) stains, we suggest using the red (PI) channel to count to avoid the impact of any autofluorescence in the green (AO) channel. Examples of trypan blue stained fixed cells are shown in FIGS. 9A-9C. High quality fixed samples have single distinct cells with <5% cell aggregation and no debris. Higher levels of aggregation will lead to elevated doublets after sequencing. When quantifying fixed cells, it is critical to avoid counting cell debris to avoid overestimating the number of cells.Centrifugation

[0318] A swinging bucket rotor should be used for all high-speed centrifugation steps in this protocol. The use of a fixed-angle rotor will lead to substantial cell loss.Maximizing Cell Recovery

[0319] It is critical to use the recommended NEB RNase Inhibitor, Murine (New England Biolabs®), in addition to the RNase Inhibitor present in the Cell Fixation kit to maximize RNA integrity and quality. It is critical to thoroughly resuspend the cells after centrifugation throughout the protocol. Resuspend by slowly and repeatedly pipetting until no clumps are visible. Ideally this should be verified with microscopy. We do not recommend wide bore pipette tips as they make it difficult to resuspend cell pellets adequately. Ensure that the 1.5 mL and 15 mL centrifuge tubes are polypropylene, as polystyrene tubes will lead to substantial sample loss.Reagent Stability

[0320] Reagents in the Cell Fixation Reagents box should not be frozen and thawed more than 3 times. The NEB RNase Inhibitor, Murine, should be stored as recommended by the manufacturer. If the kit is going to be used more than 4 times, the reagents should be aliquoted into nuclease-free 1.5 mL tubes and stored at -20°C until use. We do not recommend making single use aliquots to minimize the impact of evaporation during storage. To avoid pipetting <2 pL of RNase Inhibitor, we do not recommend preparing less than 2 reactions of Cell Storage Master Mix. In addition, DMSO should be added fresh prior to use in the protocol to a final concentration of 5%. If aliquoting is required, the master mix can be prepared without DMSO, split into aliquots, and stored at -20°C for up to a month.Storage of Fixed Samples

[0321] Fixed samples can be stored at -80°C for up to 6 months. Fixed samples should not be refrozen after thawing. When possible, we recommend splitting samples into aliquots after fixation in Section 2 Step 21. The aliquots should be >50 pL when stored in 1.5 mL tubes. If aliquots are <50 pL, we recommend store them in a PCR tube(s) / strip. We recommend making a 20 pL counting aliquot for each sample. This aliquot should be used to update sample concentrations before starting the Evercode workflow. The loss after freeze / thaw should match the sample. This will minimize the time between sample thawing and the start of the Evercode workflow.Section 1. Setup1.1. Block tubes with BSA

[0322] Although not required, blocking tubes with BSA can increase cell retention. When Protein LoBind tubes are not available, we recommend blocking tubes, especially for samples with low cell inputs or cells prone to aggregation. To block tubes: 1. Prepare a fresh 1% BSA as follows, depending on the number of samples being processed: for 1 sample: 2.6 mL nuclease-free water, 400 mL Gibco Bovine Albumin Fraction V (7.5% solution), total volume: 3 mL; for 12 samples: 31.2 mL nuclease-free water, 4.8 mL Gibco Bovine Albumin Fraction V (7.5% solution), total volume: 36 mL. 2. For each sample, fill two 1.5 mL tubes with 1.5 mL of 1% BSA and cap the tubes. 3. Invert once to fully coat the tubes. 4. Incubate the tubes for 30 minutes at room temperature. 5. Remove the 1% BSA with a P1000 and discard. 6. Remove any remaining solution from the bottom of the tube with a P200. 7. With the caps removed, air dry the tubes for 30 minutes in a biosafety cabinet at room temperature. 8. Proceed to Section 1.2 or store capped BSA-coated tubes at 4°C for up to 4 weeks.1.2 Prepare master mixes.

[0323] Master mixes should be prepared just prior to fixation. To prepare master mixes: 1. Fill a bucket with ice. Gather the following items: Prefixation buffer, Storage buffer, Fixative Solution A, Fixative Solution B, Permeabilization Solution, Fix and Perm Stop buffer, DMSO, RNase inhibitor, NEB Rnase inhibitor, Murine, Prefixation Enhancer. 2. Prepare the Cell Prefixation Master Mix in a new tube as follows: for 1 sample: Prefixation buffer (203.5 mL); RNase Inhibitor (2.75 mL), Prefixation enhancer (13.75 mL), total volume (220 mL); for 12 samples: Prefixation buffer (2.44 mL); RNase Inhibitor (33 mL), Prefixation enhancer (165mL), total volume (2.64 mL). Mix thoroughly by pipetting, carefully avoiding to create bubbles, and store on ice. 3. Prepare the Cell Fixative Master Mix in a new tube as follows: for 1 sample: Fixative Solution A (36 mL); Fixative Solution B (36 mL), total volume (72 mL); for 12 samples: Fixative Solution A (432 mL); Fixative Solution B (432 mL), total volume (864 mL). Mix thoroughly by pipetting and store on ice.

[0324] 4. Prepare the Cell Storage Master Mix in a new tube as shown in Table 44: Mix thoroughly by pipetting and store on ice. Proceed immediately to Section 2.Section 2: Fixation2.1. Cell Fixation

[0325] After the initial centrifugation to remove the buffer / medium from the single cell suspension, cells are transferred to Cell Prefixation Master Mix. Reagents are added to fix and permeabilize cells, and then to stop these reactions. Cells are resuspended in Cell Storage Master Mix and stored at -80°C or processed immediately with a downstream Evercode kit. To fix cells: 1. Cool the centrifuge with a swinging bucket rotor to 4°C. 2. Fill a bucket with ice. 3. Prepare a hemocytometer, flow cytometer, or other cell counting device. 4. Place a Mr. Frosty Freezing Container at room temperature. 5. Count the cells in the single cell suspension with a hemocytometer or alternative counting device and record the count. Keep cells on ice during counting and work quickly to minimize time on ice prior to fixation. 6. Transfer 100,000 to 1 million cells from each sample into a Protein LoBind 1.5 mL tube (or a BSA coated tube if prepared in Section 1.1). 7. Centrifuge the tubes in a swinging bucket rotor for 5-10 minutes at 200-500 x g at 4°C. Note: Use of a fixed-angle rotor in this protocol will lead to substantial cell loss. Note: Ideal centrifugation speed and duration should be determined for each sampletype to optimize retention and resuspension efficiencies. Move quickly and handle the samples gently to avoid dislodging the pellet, which will impact data quality.

[0326] 8. Slowly aspirate then discard the supernatant. 9. Fully resuspend each pellet in187.5 pL of Cell Prefixation Master Mix. 10. Pipette each sample through a cell strainer into a new 1.5 mL tube and store on ice. Note: Do not directly touch the mesh of cell strainer(s). Note: To ensure that all of the liquid passes through the strainer, press the tip of the pipette against the filter and steadily depress down the pipette plunger. All of the liquid should pass through the strainer in ~1 second. 11. Add 62.5 pL of Cell Fixative Master Mix to each tube and mix immediately by pipetting exactly 3x. 12. Incubate on ice for 10 minutes. 13. Add 20 pL of Permeabilization Solution to each tube. Immediately mix thoroughly by pipetting 3x with a P200 set to 180 pL. 14. Incubate on ice for 3 minutes. 15. Mix the Fix and Perm Stop Buffer by inverting the tube 5x. Do not vortex. 16. Add 250 pL of Fix and Perm Stop Buffer to each tube and gently pipette mix 3x. 17. Centrifuge in a swinging bucket rotor for 5-10 minutes at 200-500 x g at 4°C. 18. With a P1000 set to 500 pL, slowly aspirate then discard the supernatant. 19. Fully resuspend each pellet in 50-100 pL Cell Storage Master Mix and store on ice.

[0327] Note: Choose a resuspension volume appropriate for the experimental design and downstream Evercode kits. 20. Pipette the sample through a cell strainer into a new 1.5 mL tube and store on ice. 21. While minimizing time on ice, count the number of cells in the sample with a hemocytometer or alternative counting device and record the cell count. Note: Downstream Evercode processing can be streamlined by aliquoting samples at this step. 22. Proceed to the appropriate user guide if immediately processing samples with an Evercode kit. Otherwise, proceed to the next step. Note: If collecting and storing multiple samples over time, we recommend transferring 20 pL to a new 0.2 mL tube strip. These aliquots can be thawed and counted separately from the remaining sample for downstream processing with Evercode kits. 23. Store tubes in a Mr. Frosty Freezing Container (or equivalent device) at -80°C, according to the manufacturer’s instructions. Safe stopping point: Samples are stable for up to 6 months at -80°C. 24. The day before running the downstream Evercode kit, thaw the 20 pL aliquots in a water bath set to 37°C. Count the cells in the single cell suspension with a hemocytometer or alternative counting device and record the count. Discard any remaining sample from the counting aliquot.Centrifugation Optimization

[0328] When using Evercode Fixation kits for the first time or when testing a new sample type, we recommend optimizing centrifugation conditions. This appendix provides guidelines for optimization, suggestions for common sample types, and an example experiment to optimize centrifugation speed. Note that physical properties of cells may change after the fixation process, which requires centrifugation conditions to be optimized during fixation.

[0329] A range of centrifugation speeds should be tested to identify a speed that maximizes sample retention and permits thorough resuspension into a high quality single cell solution. Cells should be examined under a microscope before and after centrifugation to calculate cell retention and assess any aggregation or morphological changes. After determining the appropriate centrifugation conditions, we recommend using the same speed and duration throughout this and downstream Evercode User Guides.Typical Sample Retention

[0330] Across a range of samples, cell retention post-fixation typically varies between 40- 60% of the initial input. Retention is impacted by sample type, sample preparation method, centrifugation conditions, and sample handling.Speed

[0331] Increasing centrifugation speeds can improve cell retention, but high speeds can complicate the pellet resuspension and damage or even lyse cells. The optimal centrifugation speed will generally achieve a greater than 50% retention through the centrifugation step while maintaining membrane integrity. Centrifugation speed depends on cell size. Smaller cells need faster speeds, and larger cells need slower speeds.Duration

[0332] If cells are damaged by increased centrifugation speed, centrifugation duration can be adjusted to increase retention without cell damage.Temperature

[0333] For most sample types, the centrifugation should be done at 4°C. However, some sample types may require different temperatures to maximize cell viability prior to fixation. For example, isolated dendritic cells, myeloid-derived suppressor cells, and macrophages aresensitive to cold temperatures and should be processed at 25°C until the addition of the Cell Fixative Master Mix. After fixation, the final centrifugation step in this User Guide and all centrifugation steps in the Evercode User Guide should be done at 4°C to maintain cell and RNA integrity.Aggregates After Centrifugation

[0334] If the pellet cannot be resuspended back into a single cell suspension and there are aggregates where there were previously not, this is an indication that the sample may have been over centrifuged. Aggregates may also be an indication of insufficient pipette mixing. Gently resuspend the pellet by slowly and repeatedly pipetting until no clumps are visible. This can be visually inspected via microscopy. Aggregates at this stage may also be a result of the sample preparation method used. If none of the above have been successful in removing the aggregates, a filtering step may help remove aggregates or the sample preparation may require additional optimization.Debris After Centrifugation

[0335] Samples with viability of <70% may result in excessive debris in your fixed sample. Ideally, measures should be taken to optimize sample quality prior to proceeding into fixation. The Parse Biosciences applications support team can provide sample preparation optimization techniques. If a sample with minimal debris has significant debris after centrifugation, this may be an indication that the sample has lysed due to over centrifugation and / or overly aggressive resuspension. The centrifugation speed should be reduced and / or pellets should be less aggressively pipetted.Recommendations for Common Sample Types

[0336] These centrifugation conditions can be used as a starting point for common sample types. However, as samples vary, we still recommend following using the optimization protocol below. Sample type: HEK293, 3T3, and other cell lines: 200 x g, 10 min, 4 °C. Sample type: PBMCs: 200-400 x g, 10 min, 4 °C.Centrifugation Optimization Method

[0337] When using Evercode Fixation kits for the first time or when testing a new sample type, we recommend using 1-2 samples to optimize centrifugation conditions prior toprocessing samples of interest. When this is not possible, centrifugation conditions can be determined while fixing samples of interest.

[0338] FIG. 15 outlines suggested modifications to the fixation protocol to test different centrifugation conditions. This approach starts centrifugation at a low speed, but retains the supernatants after each spin. These supernatants are then centrifuged again to recover additional cells. After resuspension, each pellet should be assessed with microscopy to count cells, quantify debris, and assess aggregation. Resuspended pellets of high quality (minimal debris, minimal aggregation, and minimal evidence of cell damage) are pooled and can be used with downstream Evercode kits. If the retention is below 40-60% after pooling, we recommend contacting our applications support team for additional recommendations.

[0339] To modify the fixation protocol and optimize the centrifugation as suggested, complete all the steps as outlined in Section 1, follow steps 1-15 in Section 2. Then follow steps 16-24 as follows. 16. Centrifuge in a swinging bucket rotor for 10 minutes at 200 x g at 4°C. 17. Transfer each supernatant to a new 1.5 mL tube(s). 18. With a P200, fully resuspend each pellet in 30 pL of Cell Storage Master Mix and store on ice. 19. Repeat steps 16-18 between 2-4x, increasing the centrifugation speed by 50-100 x g each centrifugation. 20. While minimizing time on ice, count the cells and assess their quality with a hemocytometer or alternative counting device and record the cell count. 21. Calculate the retention for each centrifugation condition by comparing the number of cells input into fixation and the number of cells recovered. 22. Assess the level of debris, aggregation, and cell damage in each resuspended cell pellet. 23. Pool the high quality resuspended pellets and discard any low quality ones. Note: If the retention is below 40-60% after pooling, we recommend contacting our applications support team for additional recommendations. 24. Proceed to step 19 in Section 2.High Input Workflow Set Up

[0340] If desired, 1-4 million cells can be fixed in a single reaction. However, this requires the reagent volume to be scaled up 4x, which reduces the total number of samples that can be fixed with a 12 reaction kit to 3 samples. FIG. 16 outlines a protocol for the high input fixation workflow.Block Tubes with BSA

[0341] Although not required, blocking tubes with BSA can increase cell retention. To block tubes: 1. Prepare a fresh 1% solution. For each sample, fill two 15 mL polypropylene centrifuge tubes with 15 mL of 1% BSA and cap the tubes. 3. Invert once to fully coat the tubes. 4. Incubate the tubes for 30 minutes at room temperature. 5. Decant and discard the 1% BSA. Remove any remaining solution from the bottom of the tube with a Pl 000. 6. With the caps removed, air dry the tubes for 30 minutes in a biosafety cabinet at room temperature. 7. Proceed to prepare fixation master mixes (Prefixation master mix, fixation master mix, cell storage master mix), or store BSA-coated tubes at 4°C for up to 4 weeks.Cell Fixation

[0342] After the initial centrifugation to remove the buffer / medium from the single cell suspension, cells are transferred to Cell Prefixation Master Mix. Reagents are added to fix and permeabilize cells, and then to stop these reactions. Cells are resuspended in Cell Storage Master Mix and stored at -80°C. To fix cells: 1. Cool the centrifuge with a swinging bucket rotor to 4°C. 2. Fill a bucket with ice. 3. Prepare a hemocytometer, flow cytometer, or other cell counting device. 4. Place a Mr. Frosty Freezing Container at room temperature. 5. Count the cells in the single cell suspension with a hemocytometer or alternative counting device and record the count. Keep cells on ice during counting and work quickly to minimize time on ice prior to fixation. 6. Transfer 1-4 million cells from each sample into a 15 mL polypropylene centrifuge tube (or BSA-coated polypropylene centrifuge tube if prepared previously). 7. Centrifuge the tubes in a swinging bucket rotor for 5-10 minutes at 200-500 x g at 4°C. 8. Slowly aspirate then discard the supernatant. 9. Fully resuspend each pellet in 750 pL of Cell Prefixation Master Mix. 10. Pipette each sample through a cell strainer into a new 15 mL polypropylene centrifuge tube (or BSA-coated polypropylene centrifuge tube) with a Pl 000 and store on ice. CRITICAL! Do not directly touch the mesh of cell strainer(s). Note: To ensure that all of the liquid passes through the strainer, press the tip of the pipette against the filter and steadily depress down the pipette plunger. All of the liquid should pass through the strainer in ~1 second. 11. Add 250 pL of Cell Fixative Master Mix to each tube and mix immediately by pipetting exactly 3x with a Pl 000 set to 250 pL.

[0343] 12. Incubate on ice for 10 minutes. 13. Add 80 pL of Permeabilization Solution to each tube. Immediately mix thoroughly by pipetting 3x with a P1000 set to 250 pL. 14. Incubate on ice for 3 minutes. 15. Mix the Fix and Perm Stop Buffer by inverting the tube 5x. Do notvortex. 16. Add 1 mL of Fix and Perm Stop Buffer to each tube. Gently pipette 3x with a P1000 set to 1000 pL. 17. Centrifuge in a swinging bucket rotor for 5-10 minutes at 200-500 x g at 4°C. 18. Remove and discard the supernatant. 19. Fully resuspend each pellet in 150 pL Cell Storage Master Mix and store on ice. 20. Pipette each sample through a cell strainer into a new 1.5 mL tube with a P1000 and store on ice. 21. While minimizing time on ice, count the number of cells in the sample with a hemocytometer or alternative counting device and record the cell count. Note: Downstream Evercode processing can be streamlined by aliquoting samples at this step. 22. Proceed to the appropriate user guide if immediately processing samples with an Evercode kit. Otherwise, proceed to the next step. 23. Store tubes in a Mr. Frosty Freezing Container (or equivalent device) at -80°C, according to the manufacturer’s instructions. Safe stopping point: Samples are stable for up to 6 months at -80°C.Example 3, Exemplary fixation workflow with low cell input (e.g., less than 100k cells).

[0344] From a single cell suspension, the Evercode Low Input Fixation kits generate fixed and permeabilized cells / nuclei ready for use in the appropriate Evercode assays. This protocol can be used to fix cells or nuclei for various single-cell applications, including single-cell RNA- seq (e.g., Evercode WT) or immune profiling applications (e.g., BCR profiling), e.g., using mammalian cells such as human or mouse cells.

[0345] This workflow is designed to efficiently process between 10,000 and 100,000 cells or nuclei, accommodating up to 12 samples or as many as 96 samples simultaneously. The fixation protocol preserves cell structure, prevents RNA degradation, and locks RNA inside the cells, essential for downstream processing with Evercode's split-pool combinatorial barcoding technology (see, e.g., FIGS. 1-7). The workflow allows for high levels of cell retention with low numbers of inputted cells. Fixed samples are stable for up to 4 months at - 80°C, providing flexibility by decoupling sample collection from library preparation. This allows samples to be stored and batched post-fixation, enabling simultaneous library preparation and minimizing batch effects. The workflow facilitates parallel fixation of multiple samples, streamlining the process when handling up to 96 samples at a time.

[0346] FIG. 17 provides an overview of the low input fixation workflow. Between 10,000 and 100,000 cells / nuclei can be fixed in a single reaction. After fixation, cells / nuclei can either be stored at 80°C or immediately proceed with capture and barcoding.

[0347] Table 45 provides details of the total and hands-on time required for the cell / nuclei fixation workflow.Sample Input

[0348] This protocol begins with a previously prepared suspension of single cells / nuclei. We recommend suspensions with <5% aggregation / debris. If the cells / nuclei were previously frozen, ensure the suspension is completely thawed and in suspension before beginning fixation. We recommend minimizing the length of time samples are stored on ice prior to fixation, as it can negatively impact results. Between 10,000 and 100,000 cells / nuclei can be fixed in a single reaction. Exceeding 100,000 cells / nuclei in a single fixation will result in substantially elevated doublet rates. The minimum input into fixation should also be determined based on how the samples will be processed downstream. Table 46 provides guidance on the post-fixation concentrations needed for downstream kits. However, more or less sample input may be required depending on the exact experimental design. Note that some cells / nuclei will be lost when freezing and thawing fixed samples, typically between 5-15%. Also consider the average bead binding retention being 65% for small cells / nuclei and 80% for large cells / nuclei. These factors should be taken into account when determining how much sample input is needed for fixation. There is one centrifugation step in this workflow. In this step cell loss will vary depending on cell type and spin speeds. We recommend optimizing centrifugation speed to minimize cell loss. See "Centrifugation" section below, for more details.Table 46. Cells / Nuclei ConcentrationsAvoiding RNase Contamination

[0349] Standard precautions should be taken to avoid introducing RNases into samples or reagents throughout the workflow. Always wear proper laboratory gloves and use aseptic technique. Although RNases are not inactivated by ethanol or isopropanol, they are inactivated by products such as RNaseZap RNase Decontamination Solution (Thermo Fisher Scientific). These can be sprayed on benchtops and pipettes. Filtered pipette tips should be used to reduce RNase contamination from pipettes. Filtered pipette tips should be used to reduce RNase contamination from pipettes.Cell / Nuclei Strainers

[0350] To maximize cells / nuclei retention with cell strainers, press the pipette tip directly against the mesh. Ensure ample pressure is applied to hold contact between the tip and the strainer to force liquid through in ~1 second. A strainer with an appropriately sized mesh should be used throughout the protocol. Although 30-40 pm is appropriate for most cells / nuclei, the mesh size should be chosen based on your sample type.Cell / Nuclei Counting and Quality Assessment

[0351] We recommend a hemocytometer for counting, but alternative counting devices can also be used. If possible, validate counts from alternative devices to a hemocytometer when first using Evercode Fixation kits. When first using Evercode Fixation kits, we suggest saving images at each counting step. After fixation, the cells / nuclei are permeabilized and should appear dead with viability stains. We recommend Trypan Blue for all counting up until the bead binding step. After the beads are bound, we recommend using fluorescent staining such as Acridine Orange / Propidium Iodide (AO / PI) or Acridine Orange / DAPI (AO / DAPI) (see, FIGS. 18A-18B).

[0352] Examples of stained fixed cells are shown in FIGS. 9A-9C. High quality fixed samples have single distinct cells / nuclei with <5% aggregation and no debris. Higher levels of aggregation will lead to elevated doublets after sequencing and may indicate a poor quality cells / nuclei isolation. When quantifying fixed cells / nuclei, it is critical to avoid counting debris to avoid overestimating the number of cells / nuclei.Centrifugation Maximizing Cells / Nuclei Recovery Debris

[0353] There is only one centrifugation step in this protocol. A range of centrifugation speeds and durations are given rather than a single speed. When using Evercode Low Input Fixation kits for the first time or when testing a new sample type, we recommend optimizing centrifugation conditions in 1.5 mL tubes before using the plate-based workflows. A swinging bucket rotor should be used for the high-speed centrifugation step in this protocol. The use of a fixed-angle rotor will lead to substantial cells / nuclei loss.Maximizing Cell / Nuclei Recovery

[0354] It is critical to thoroughly resuspend the cells / nuclei after centrifugation. Resuspend by slowly and repeatedly pipetting up and down until no clumps are visible. Ideally this should be verified with microscopy. To minimize cells / nuclei loss from cells / nuclei adherence to tubes, carefully pipette up and down along the bottom and sides of tubes. We do not recommend wide bore pipette tips as they make it difficult to resuspend cells / nuclei pellets adequately. Ensure that the 0.2 mL centrifuge tubes / plates are polypropylene, as polystyrene tubes / plates will lead to substantial sample loss.Reagent Stability

[0355] With the exception of the Cell / Nuclei Binding Beads, reagents in the Fixation Reagents box can be frozen and thawed up to 3 times. It is critical to never freeze the beads or vortex them for an extended period of time. If the kit is going to be used more than 4 times, the reagents should be aliquoted into nuclease-free 1.5 mL tubes and stored at -20°C until use. We do not recommend making single use aliquots to minimize the impact of evaporation during storage. Reagent master mixes should be made fresh and used the same day.Storage of Fixed Samples

[0356] Fixed samples can be stored at -80°C for up to 4 months. Fixed samples should not be refrozen after thawing.Section 1. 12 Reactions - Cell / Nuclei FixationLow Input Evercode Cell / Nuclei Fixation Kits Parts Lists (for 12 Reactions)

[0357] The Evercode Low Input Cell Fixation, 12 reactions workflow requires Cell Fixation Reagents, Cell Prefixation Enhancer, Cell Binding Reagents, Cell Binding Beads boxes, and Plate Strainer boxes, and the Evercode Low Input Nuclei Fixation, 12 reactions workflow requires Nuclei Fixation Reagents, Nuclei Prefixation Enhancer, Nuclei Binding Reagents, Nuclei Binding Beads boxes, and Plate Strainer boxes. The Plate Strainer box should have an appropriate mesh size for the cell or nucleus type being fixed.User Supplied Equipment and Reagents

[0358] The following materials and equipment are required to perform the protocol but are not provided within the kit. This list does not include standard laboratory equipment, such as freezers.1.1: 12 Reactions - Prepare Master Mixes

[0359] Prior to initiating the cell / nuclei fixation process, master mixes are prepared. Master mixes are tailored for each sample type (i.e., cells or nuclei). The Low Input Evercode Cell Fixation kit necessitates the preparation of 3 distinct master mixes: i) Cell Prefixation Master Mix, ii) Cell Fixative Master Mix, and iii) Cell Fix and Perm Stop Master Mix. The Cell Fixation master mixes should be prepared immediately prior to the cell fixation process. TheLow Input Evercode Nuclei Fixation kit necessitates the preparation of 2 distinct master mixes: i) Nuclei Prefixation Master Mix, and ii) Nuclei Fix and Perm Stop Master Mix. The Nuclei Fixation master mixes should be prepared immediately prior to the nuclei fixation process.1.1.1: Prepare Cell Master Mix

[0360] To prepare master mixes for Cell Fixation: 1. Fill a bucket with ice. Gather the items shown in Table 51 and handle as indicated.

[0361] Prepare the Cell Prefixation Master Mix in a new tube as shown in Table 52. Mix thoroughly by pipetting and store on ice.

[0362] Note: To avoid pipetting <2 pL of RNase Inhibitor, we do not recommend preparing fewer than two reactions of Cell Prefixation Master Mix. Note: Reagents in the Cell Fixation Reagents box can be frozen and thawed 3 times. If the kit will be used more than 4 times, aliquots should be made. See Reagent Stability section for details. 3. Prepare the Cell Fixative Master Mix in a new tube as shown in Table 53. Mix thoroughly by pipetting and store on ice.

[0363] 4. Prepare the Cell Fix and Perm Stop Master Mix in a new tube as shown in Table54. Mix thoroughly by pipetting and store on ice.

[0364] 5 With the Plate Strainers still in the plastic sleeve, cut along the green plastic with sterile scissors, a razor blade, or a scalpel that has been cleaned with RNaseZap. Cut the strip according to the number of wells intended to use. Note: Do not directly touch the mesh of Plate Strainer(s) to anything prior to straining. Note: After opening their plastic sleeves, Plate Strainers should be stored in their original plastic sleeve until use and used on the same day they were opened. 6. Proceed immediately to Section 1.2.1.1.2: Prepare Nuclei Master Mix

[0365] To prepare master mixes for Nuclei Fixation: 1. Fill a bucket with ice. Gather the items shown in Table 55 and handle as indicated.

[0366] Prepare the Nuclei Prefixation Master Mix in a new tube as shown in Table 56. Mix thoroughly by pipetting and store on ice.

[0367] Note: To avoid pipetting <2 pL of RNase Inhibitor, we do not recommend preparing less than 2 reactions of Nuclei Prefixation Master Mix. See Reagent Stability section for additional details. Note: Reagents in the Nuclei Fixation Reagents box can be frozen and thawed 3 times. If the kit will be used more than 4 times, aliquots should be made. See Reagent Stability section for details. 3. Prepare the Nuclei Fix and Perm Stop Master Mix in a new tube as shown in Table 57. Mix thoroughly by pipetting and store on ice.

[0368] 4. With the Plate Strainers still in the plastic sleeve, cut along the green plastic with sterile scissors, a razor blade, or a scalpel that has been cleaned with RNaseZap. Cut the strip according to the number of wells intended to use. Note: Do not directly touch the mesh of Plate Strainer(s) to anything prior to straining. Note: After opening their plastic sleeves, Plate Strainers should be stored in their original plastic sleeve until use and used on the same day they were opened. 6. Proceed immediately to Section 1.2.1.2: 12 Reactions - Cell / Nuclei Fixation

[0369] After the initial centrifugation to remove the buffer / medium from the single cell suspension, Prefixation Master Mix is added to the cells / nuclei. Reagents are added to fix and permeabilize cells / nuclei, and then stop these reactions. Fixed cells / nuclei are stored at -80°C.

[0370] To fix cells / nuclei: 1. Cool the centrifuge with a swinging bucket rotor to 4°C. 2. Fill a bucket with ice. 3. Prepare a hemocytometer, flow cytometer, or other cell / nuclei counting device. 4. Count the cells / nuclei in the single cell suspension with a hemocytometer or alternative counting device and record the count. Keep cells / nuclei on ice during counting and work quickly to minimize time on ice prior to fixation. 5. Transfer 10,000 to 100,000 cells / nuclei from each sample into 0.2 mL tube(s) / plate. Seal the tube(s) / plate with caps or an adhesive seal. 6. Centrifuge the 0.2 mL tube(s) / plate in a swinging bucket rotor for 5-10 minutes at 200-500 x g at 4°C. Note: A small cell / nuclei loss should be expected after centrifugation. Note: Use of a fixed-angle rotor in this protocol will lead to substantial cells / nuclei loss. Ideal centrifugation speed and duration should be determined for each sample type to optimize retention and resuspension efficiencies. Move quickly and handle the samples gently to avoid dislodging the pellet, which will impact data quality.

[0371] 7. Slowly aspirate then discard the supernatant, leaving no more than 20 pL of supernatant. 8. Fully resuspend each pellet in 60 pL of Cell / Nuclei Prefixation Master Mix. 9. Apply a Plate Strainer to a new 0.2 mL tube(s) / plate by peeling off the backing, carefullyaligning over the wells, and placing on the surface of the tube(s) / plate. Note: The Plate Strainer fits a 96 well plate. It will need to be cut when processing 12 samples. 10. Without touching the mesh, fully adhere the Plate Strainer by pressing a pipette tip or plate sealer cleaned with RNaseZap along the green plastic on the edges and between the wells of the tube(s) / plate. 11. Pipette 75 pL of each sample through the strainer into the new 0.2 mL tube(s) / plate and store on ice. Note: Do not directly touch the mesh of the strainer(s) with anything except the pipette tip. Note: To ensure that all of the liquid passes through the strainer, firmly press the tip of the pipette against the filter and steadily depress down the pipette plunger. All of the liquid should pass through the strainer in ~1 second.

[0372] 12. Carefully peel off the Plate Strainer and discard. 13. Add 25 pL of Cell FixativeMaster Mix / Fixative Solution to each well and mix immediately by pipetting exactly 3x. Note: Do not perform additional mixing at this step. 14. Incubate on ice for 10 minutes. 15. Add 8 pL of Cell / Nuclei Permeabilization Solution to each tube. Immediately mix thoroughly by pipetting 3x with a P200 set to 80 pL. 16. Incubate on ice for 3 minutes. 17. Mix the Cell / Nuclei Fix and Perm Stop Master Mix by inverting the tube 5x. Do not vortex. 18. Add 110.4 pL of Cell / Nuclei Fix and Perm Stop Master Mix to each well. Gently pipette 3x. 19. Proceed to Section 1.3: 12 Reactions - Cells / Nuclei Capture, if immediately processing samples with an Evercode WT kit. Otherwise, proceed to the next step to freeze and save the fixed cells / nuclei. 20. Place the samples in a room temperature styrofoam cooler, close the lid, and store at -80°C to slowly cool the samples. Note: Storing samples directly in the freezer without controlled cooling may lead to cells / nuclei damage and compromise data quality. Safe stopping point: Samples are stable for up to 4 months at -80°C.1.3: 12 Reactions - Cells / Nuclei Capture

[0373] After fixation, cells / nuclei need to be captured using magnetic beads and resuspended in Storage Buffer prior to barcoding. If fixed samples were stored at -80C, they will need to be thawed before the capture step. It is recommended to count the captured cells / nuclei using fluorescent-based dyes on an automated cell counter or trypan blue on a hemocytometer prior to input into barcoding. Alternatively, you may extrapolate the remaining cells / nuclei based on the previous count (Section 1.2, Step 4) and assume 65% retention for small cells / nuclei and 80% retention for large cells / nuclei.

[0374] To capture cells / nuclei: 1. Fill a bucket with ice. Gather the items and handle as indicated in Table 58.

[0375] 2. Gently pulse-vortex Cell / Nuclei Binding Beads until resuspended. Add the appropriate volume of Cell / Nuclei Binding Beads to a new 0.2 mL PCR tube as shown in Table 59, depending on the number of samples being processed:

[0376] 3. Place the tube on the magnetic rack for 0.2 mL PCR tubes until the solution clears(~2 minutes). 4. Remove and discard the supernatant. 5. Remove the tube from the magnetic rack and fully resuspend the bead pellet in the appropriate volume of Bead Wash Buffer (Table 60). Note: Ensure no beads are stuck to the sides of the 0.2 mL tube.

[0377] 6. Place the tube on the magnetic rack for 0.2 mL PCR tubes until the solution clears(~2 minutes). 7. Remove and discard the supernatant. 8. Repeat steps 4-6 twice for a total of 3 washes. 9. Remove the tube from the magnetic rack. Fully resuspend the pellet in the appropriate volume of Cell / Nuclei Storage Buffer.

[0378] 10. Remove the plate of fixed cells / nuclei from -80 °C storage. Set thermocycler to the protocol shown in Table 62.

[0379] 11. To thaw, place the tube(s) / plate of frozen cells / nuclei in the thermocycler and start the program. 12. Once the thaw protocol has finished, check that all wells are fully thawed. If ice remains, place the tube(s) / plate back into the thermocycler for another minute at 37°C. Once fully thawed, place the tube(s) / plate into a PCR plate holder, remove the plate seal, and store on ice. 13. To capture cells / nuclei, add Cell / Nuclei Binding Beads in the Cell / Nuclei Storage Buffer to each fixed sample according to Table 63. Discard unused beads. Note: With a pipette mix the beads thoroughly to fully resuspend them before using for cell capture.

[0380] 14. With a P200 pipette set to 120 pL, pipette 3x to ensure beads are fully suspended in cell / nuclei samples. 15. Place the tube(s) on a 0.2 mL tube magnet or the plate on a plate magnet and bind at room temperature for 5 minutes. 16. With a P200 pipette set to 200pL, remove supernatant from each sample, being careful not to disturb the pellet. Note: Move quickly to resuspend beads at this step to minimize time beads are out of liquid (<5 minutes). 17. Vigorously resuspend cells / nuclei in the Cell / Nuclei Storage Buffer and mix well. Table 64 indicates the minimum volume required for resuspension. Note: Resuspending the beads in less than the minimum volume required for resuspension will harm the barcoding chemistry and result in a loss of transcript detection. Note: If you have <30,000 cells / nuclei and prefer not to count, proceed assuming 65% retention for small cells / nuclei and 80% retention for large cells / nuclei. Note that these retention assumptions are an estimate and may result in under / overloading the barcoding plate. Note: Storage buffer resuspension volumes indicated in Table 64 are the minimum volumes needed to resuspend samples. Depending on sample concentration, further dilution prior to proceeding with barcoding is likely required. 18. Proceed to cell / nuclei counting and / or to Evercode Whole Transcriptome User Guide Section 1.1 Barcoding.Section 2. 96 Reactions - Cell / Nuclei FixationLow Input Evercode Cell Fixation Kit Part List (for 96 Reactions)

[0381] The High Throughput Evercode Low Input Cell Fixation kit requires Cell Fixation Reagents, Cell Prefixation Reagents, Cell Binding Reagents, Cell Binding Beads, and Plate Strainer boxes. The Plate Strainer box should have an appropriate mesh size for the cell type being fixed. The Evercode Low Input Nuclei Fixation, 96 reactions workflow requires Nuclei Fixation Reagents, Nuclei Prefixation Enhancer, Nuclei Binding Reagents, Nuclei Binding Beads boxes, and Plate Strainer boxes. The Plate Strainer box should have an appropriate mesh size for the nuclei type being fixed.User Supplied Equipment and Reagents

[0382] The following materials and equipment are required to perform the protocol but are not provided within the kit. This list does not include standard laboratory equipment, such as freezers.2.1: 96 Reactions - Prepare Master Mixes

[0383] Prior to initiating the cell / nuclei fixation process, master mixes are prepared. Master mixes are tailored for each sample type (i.e., cells or nuclei). The Low Input Evercode Cell Fixation kit necessitates the preparation of 3 distinct master mixes: i) Cell Prefixation Master Mix, ii) Cell Fixative Master Mix, and iii) Cell Fix and Perm Stop Master Mix. The Cell Fixation master mixes should be prepared immediately prior to the cell fixation process. The Low Input Evercode Nuclei Fixation kit necessitates the preparation of 2 distinct master mixes:i) Nuclei Prefixation Master Mix, and ii) Nuclei Fix and Perm Stop Master Mix. The Nuclei Fixation master mixes should be prepared immediately prior to the nuclei fixation process.2.1.1: Prepare Cell Master Mix

[0384] To prepare master mixes for Cell Fixation: 1. Fill a bucket with ice. Gather the items shown in Table 69 and handle as indicated.

[0385] Prepare the Cell Prefixation Master Mix in a new tube as shown in Table 70. Mix thoroughly by pipetting and store on ice.

[0386] Note: To avoid pipetting <2 pL of RNase Inhibitor, we do not recommend preparing less than 2 reactions of Cell Prefixation Master Mix. See Reagent Stability section for additional details. Note: Reagents in the Cell Fixation Reagents box can be frozen and thawed 3 times. If the kit will be used more than 4 times, aliquots should be made. See Reagent Stability section for details.

[0387] 3. Prepare the Cell Fixative Master Mix in a new tube as shown in Table 71. Mix thoroughly by pipetting and store on ice.

[0388] 4. Prepare the Cell Fix and Perm Stop Master Mix in a new tube as shown in Table72. Mix thoroughly by pipetting and store on ice.

[0389] 5. With the Plate Strainers still in the plastic sleeve, cut along the green plastic with sterile scissors, a razor blade, or a scalpel that has been cleaned with RNaseZap. Cut the strip according to the number of wells intended to use. Note: Do not directly touch the mesh of Plate Strainer(s) to anything prior to straining. Note: After opening their plastic sleeves, Plate Strainers should be stored in their original plastic sleeve until use and used on the same day they were opened. 6. Proceed immediately to Section 2.2.2.1.2: Prepare Nuclei Master Mix

[0390] To prepare master mixes for Nuclei Fixation: 1. Fill a bucket with ice. Gather the items shown in Table 73 and handle as indicated.

[0391] Prepare the Nuclei Prefixation Master Mix in a new tube as shown in Table 74. Mix thoroughly by pipetting and store on ice.

[0392] Note: To avoid pipetting <2 pL of RNase Inhibitor, we do not recommend preparing less than 2 reactions of Nuclei Prefixation Master Mix. See Reagent Stability section foradditional details. Note: Reagents in the Nuclei Fixation Reagents box can be frozen and thawed 3 times. If the kit will be used more than 4 times, aliquots should be made. See Reagent Stability section for details.

[0393] 3. Prepare the Nuclei Fix and Perm Stop Master Mix in a new tube as shown in Table75. Mix thoroughly by pipetting and store on ice.

[0394] 4. With the Plate Strainers still in the plastic sleeve, cut along the green plastic with sterile scissors, a razor blade, or a scalpel that has been cleaned with RNaseZap. Cut the strip according to the number of wells intended to use. Note: Do not directly touch the mesh of Plate Strainer(s) to anything prior to straining. Note: After opening their plastic sleeves, Plate Strainers should be stored in their original plastic sleeve until use and used on the same day they were opened. 6. Proceed immediately to Section 2.2.2.2: 96 Reactions - Cell / Nuclei Fixation

[0395] After the initial centrifugation to remove the buffer / medium from the single cell suspension, cells / nuclei are transferred to Prefixation Master Mix. Reagents are added to fix and permeabilize cells / nuclei, and then stop these reactions. Fixed cells / nuclei are stored at - 80°C for up to 4 months.

[0396] To fix cells / nuclei: 1. Cool the centrifuge with a swinging bucket rotor to 4°C. 2. Fill a bucket with ice. 3. Prepare a hemocytometer, flow cytometer, or other cell / nuclei counting device. 4. Count the cells / nuclei in the single cell suspension with a hemocytometer or alternative counting device and record the count. Keep cells / nuclei on ice during counting and work quickly to minimize time on ice prior to fixation. 5. With the plate on ice, transfer 10,000 to 100,000 cells / nuclei from each sample into the wells of polypropylene, nuclease-free PCR plate. 6. Seal the plate with an adhesive seal. 7. Centrifuge the plate in a swinging bucket rotor for 5-10 minutes at 200-500 x g at 4°C. Note: A small cell / nuclei loss should be expected aftercentrifugation. Note: Use of a fixed-angle rotor in this protocol will lead to substantial cells / nuclei loss. Ideal centrifugation speed and duration should be determined for each sample type to optimize retention and resuspension efficiencies. Note: Move quickly and handle the samples gently to avoid dislodging the pellet, which will impact data quality. 8. Remove the plate from the centrifuge, remove the plate seal, and store on ice. 9. With a multichannel P200, slowly aspirate and discard all but ~20 pL of supernatant from each well. Keep the pipette tips along the side of the wells to avoid disturbing the pellets. Note: Tilting the plate 90 degrees makes it easier to visualize removal of residual supernatant.

[0397] 11. Transfer the Cell / Nuclei Prefixation Master Mix to a new basin with a pipette.12. In order to prepare to strain the prefix samples, apply the Plate Strainer to a new PCR plate, carefully aligning over the wells. Without touching the mesh, fully adhere the Plate Strainer by pressing a pipette tip or plate sealer cleaned with RNaseZap along the green plastic on the edges and between the wells of the plate. Keep the plate on ice. 13. Using a multichannel P200 pipette, fully resuspend each pellet in 60 pL of Cell / Nuclei Prefixation Master Mix. Using the same set of tips, strain 75 pL of the resuspended cells into the corresponding wells of the new PCR plate from step 12. 14. Repeat Step 13 for the next row until all samples are resuspended and strained. Keep strained cells on ice. Note: Do not directly touch the mesh of Plate Strainer(s) to anything prior to straining. Note: Plate Strainer(s) will need to be cut if processing fewer than 96 samples. Note: To ensure that all of the liquid passes through the strainer, firmly press the tip of the pipette against the filter and steadily depress down the pipette plunger. All of the liquid should pass through the strainer in ~1 second.

[0398] 15. Tap the plate 3x on the benchtop to move liquid to the bottom of the wells. Check to ensure there are no bubbles in the bottom of the wells. 16. Carefully peel off the Plate Strainer and discard. 17. Transfer the Cell / nuclei Fixative Master Mix / Fixative Solution to a basin with a pipette (see Table 76).

[0399] 18. With the plate on ice, add 25 pL of Cell Fixative Master Mix / Fixative Solution to each well and mix immediately by pipetting exactly 3x. 19. Incubate on ice for 10 minutes.Note: Start the timer after adding Cell Fixative Master Mix / Fixative Solution to the first row of the plate. 20. Transfer the Cell / Nuclei Permeabilization Solution to a new basin with a pipette as indicated in Table 77.

[0400] 21. With a P20 multichannel pipette, add 8 pL of Cell / Nuclei PermeabilizationSolution to each well with a multichannel P20 and mix immediately by pipetting 3x with a multichannel P200 set to 80 pL. 22. Repeat Step 21 for each row until all samples are permeabilized. 23. Incubate on ice for 3 minutes. Note: Start the timer after adding Permeabilization Solution to the first row of the plate. This incubation can be extended by 2 additional minutes and up to a total of 5 minutes without negatively impacting performance. 24. Transfer the Cell / Nuclei Fix and Perm Stop Master Mix to a new basin with a pipette as indicated in Table 78.

[0401] 25. With the plate on ice, add 110.4 pL of Cell / Nuclei Fix and Perm Stop MasterMix to each well and mix immediately by gently pipetting 3x with a multichannel P200 set to 100 pL. 26. Proceed to Section 2.3: 96 Reactions - Cell / Nuclei Capture if immediately processing samples with an Evercode Whole Transcriptome (WT) kit. Otherwise, proceed to the next step. 27. Seal the PCR plate with a seal that can withstand storage at -80°C. Note: Many clear plastic seals are not designed for storage at -80°C, so we recommend using foil plate seals. 28. Place the samples in a room temperature styrofoam cooler, close the lid, and store at -80°C to slowly cool the samples. Note: Storing samples directly in the freezer without controlled cooling may lead to cells / nuclei damage and compromise data quality. Safe stopping point: Samples are stable for up to 4 months at -80°C.2.3: 96 Reactions - Cell / Nuclei Capture

[0402] After fixation, cells / nuclei need to be captured using magnetic beads and resuspended in Storage Buffer prior to barcoding. If fixed samples were stored at -80 °C, they will need to be thawed before the capture step. Capture of fixed cells / nuclei is performed using magnetic beads. It is recommended to count the captured cells / nuclei using fluorescent-based dyes on an automated cell counter or trypan blue on a hemocytometer prior to input into barcoding. Alternatively, you may extrapolate the remaining cells / nuclei based on the previous count (Section 2.2, Step 4) and assume 65% retention for small cells / nuclei and 80% retention for large cells / nuclei.

[0403] To capture cells / nuclei: 1. Fill a bucket with ice. Gather the items and handle as indicated in Table 79.

[0404] 2. Gently pulse-vortex Cell / Nuclei Binding Beads until resuspended. Add the appropriate volume of Cell / Nuclei Binding Beads to a new 1.5 mL tube as indicated in Table 80, depending on the number of samples being processed:

[0405] 3. Place the tube on the magnetic rack for 1.5 mL tubes until the solution clears (~2 minutes). 4. Remove and discard the supernatant. 5. Remove the tube from the magnetic rack and fully resuspend the bead pellet in the appropriate volume of Bead Wash Buffer (Table 81). Note: Ensure no bea...

Claims

CLAIMS:

1. A method of uniquely labeling RNA molecules within a plurality of B cells, the method comprising:(a) fixing and permeabilizing a plurality of B cells;(b) dividing the plurality of B cells into a first plurality of aliquots, wherein each aliquot comprises more than one cell;(c) generating complementary DNA (cDNA ) molecules within the B cells of the first plurality of aliquots, wherein the RNA molecules are reverse transcribed using reverse transcription (RT) primers each comprising: (i) a poly(T) sequence or a random sequence; and (ii) an RT barcode sequence, wherein the RT barcode sequences present within the RT primers are specific to each aliquot;(d) pooling the B cells from the first plurality of aliquots;(e) tagging the cDNA molecules within the pooled B cells with one or more nucleic acid tags by performing steps (e)(7) through (e)(zzz) one or more times:(z) dividing the pooled B cells into an additional plurality of aliquots;(zz) coupling nucleic acid tags to the cDNA molecules within the B cells of the additional plurality of aliquots, thereby generating a plurality of tagged cDNA molecules within each cell, wherein each nucleic acid tag comprises a tag barcode sequence, and wherein the tag barcode sequences present within the nucleic acid tags are specific to each aliquot;(Hi combining the B cells from the additional plurality of aliquots;(f) dividing the combined B into a plurality of sublibraries;(g) lysing the B cells in one or more sublibrary of the plurality of sublibraries to release the plurality of tagged cDNA molecules from each cell and produce a lysate comprising the released tagged cDNA molecules;(h) isolating the released tagged cDNA molecules from the lysate;(i) preparing separate sequencing libraries from the released tagged cDNA molecules for: (z) the whole transcriptome or a subset thereof, and (zz) BCR- encoding transcripts within the transcriptome.

2. The method of claim 1, wherein preparing the sequencing libraries comprises amplifying the released tagged cDNA molecules during one or more rounds of amplification, wherein at least a portion of the primers used to amplify the released tagged cDNA molecules in one or more of the rounds of amplification comprise an index sequence, and wherein the index sequences present within the at least a portion of the primers are specific to each sublibrary.

3. The method of claim 1 or claim 2, wherein preparing the sequencing library for the BCR-encoding transcripts comprises enriching the released tagged cDNA molecules for cDNA sequences encoding BCRs.

4. The method of claim 3, wherein the released tagged cDNA molecules are enriched for cDNA sequences encoding BCRs by hybrid capture.

5. The method of claim 4, wherein the hybrid capture is performed using probes targeting any one or more of the sequences shown in Table 13 and / or Table 14 and / or shown as SEQ ID NOS: 750-944 and / or 945-1121.

6. The method of claim 5, wherein the hybrid capture is performed using probes targeting all of the sequences shown in Table 13 and / or all of the sequences shown in Table 14 and / or shown as SEQ ID NOS: 750-944 and / or 945-1121.

7. The method of claim 5 or 6, wherein the hybrid capture is performed using probes targeting at least about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the sequences shown in Table 13 and / or Table 14 and / or shown as SEQ ID NOS: 750-944 and / or 945-1121.

8. The method of any one of claims 5-7, wherein the hybrid capture is performed using probes comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology or complementarity to at least about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequences shown in Table 13 and / or Table 14 and / or shown as SEQ ID NOS: 750-944 and / or 945-1121.

9. The method of any one of claims 1-8, wherein the B cells comprise human B cells, and wherein the hybrid capture is performed using probes targeting any one or more of the sequences shown in Table 13 and / or shown as SEQ ID NOS: 750-944, or targeting a subsequence of any of the sequences shown in Table 13 and / or shown as SEQ ID NOS: 750- 944, or comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology or complementarity to at least about 50%, 55%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequences shown in Table 13 and / or shown as SEQ ID NOS: 750-944.

10. The method of any one of claims 1-8, wherein the B cells comprise mouse B cells, and wherein the hybrid capture is performed using probes targeting sequences shown in Table 14 and / or targeting sequences shown as SEQ ID NOS: 945-1121, or targeting a subsequence of any of the sequences shown in Table 14 and / or shown as SEQ ID NOS: 945- 1121, or comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology or complementarity to at least about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequences shown in Table 14 and / or shown as SEQ ID NOS: 945-1121.

11. The method of any one of claims 1-8, wherein the B cells comprise chimeric human-mouse B cells, and wherein the hybrid capture is performed using probes targeting sequences shown in Tables 13 and sequences shown in Table 14 and / or targeting sequences shown as SEQ ID NOS: 750-944 and sequences shown as SEQ ID NOS: 945-1121, probes targeting any one or more of the sequences shown in Table 13 and any one or more of the sequences shown in Table 14 and / or targeting any one or more of SEQ ID NOS: 750-944 and any one or more of SEQ ID NOS: 945-1121, probes targeting a sequence or subsequence of any one or more of the sequences shown in Table 13 and targeting a sequence or subsequence of any one or more of the sequences shown in Table 14, and / or targeting a sequence or subsequence of any one or more of SEQ ID NOS: 750-944 and targeting a sequence or subsequence of any one or more of the SEQ ID NOS: 945-1121, and / or probes comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology or complementarity to at least about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequences shown in Table 13 and / or shown as SEQ ID NOS: 750-944, and comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology or complementarity to at least about 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequences shown in Table 14 and / or shown as SEQ ID NOS: 945-1121.

12. The method of any one of claims 4-11, wherein the hybrid capture is performed using one or more probes targeting one or more genes selected from the group IGHA1, IGHA2, IGHD, IGHD1-1, IGHD1-7, IGHD1-14, IGHD1-20, IGHD1-26, IGHD1OR15-1A, IGHD1OR15-1B, IGHD2-2, IGHD2-8, IGHD2-15, IGHD2-21, IGHD2OR15-2A, IGHD2OR15-2B, IGHD3-3, IGHD3-9, IGHD3-10, IGHD3-16, IGHD3-22, IGHD3OR15-3A,IGHD3OR15-3B, IGHD4-4, IGHD4-11, IGHD4-17, IGHD4-23, IGHD4OR15-4A,IGHD4OR15-4B, IGHD5-5, IGHD5-12, IGHD5-18, IGHD5-24, IGHD5OR15-5A,IGHD5OR15-5B, IGHD6-6, IGHD6-13, IGHD6-19, IGHD6-25, IGHD7-27, IGHE, IGHG1, IGHG2, IGHG3, IGHG4, IGHGP, IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, IGHJ6, IGHM, IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18, IGHV1-24, IGHV1-38-4, IGHV1-45, IGHV1-46, IGHV1-58, IGHV1-69, IGHV1-69-2, IGHV1-69D, IGHV10R15-1, IGHV1OR15-5, IGHV1OR15-9, IGHV10R21-1, IGHV2-5, IGHV2-26, IGHV2-70, IGHV2-70D, IGHV2OR16-5, IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-16, IGHV3- 20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-30-3, IGHV3-30-5, IGHV3-33, IGHV3-35, IGHV3-38, IGHV3-38-3, IGHV3-43, IGHV3-43D, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-64D, IGHV3-66, IGHV3-72, IGHV3-73, IGHV3-74, IGHV3OR16-8, IGHV3OR16-9, IGHV3OR16-10, IGHV3OR16-12, IGHV3OR16-13, IGHV3OR16-17, IGHV4-4, IGHV4-28, IGHV4-30-1, IGHV4-30-2, IGHV4-30-4, IGHV4-31, IGHV4-34, IGHV4-38-2, IGHV4-39, IGHV4-59, IGHV4-61, IGHV4OR15-8, IGHV5-10-1, IGHV5-51, IGHV6-1, IGHV7-4-1, IGHV7-81, IGHV8-51-1, IGKC, IGKJ1, IGKJ2, IGKJ3, IGKJ4, IGKJ5, IGKV1-5, IGKV1-6, IGKV1-8, IGKV1-9, IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37, IGKV1-39, IGKV1D-8, IGKV1D-12, IGKV1D-13, IGKV1D-16, IGKV1D-17, IGKV1D-33, IGKV1D-37, IGKV1D-39, IGKV1D- 42, IGKV1D-43, IGKV1OR2-0, IGKV1OR2-108, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-40, IGKV2D-24, IGKV2D-26, IGKV2D-28, IGKV2D-29, IGKV2D-30, IGKV2D-40, IGKV3-7, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-7, IGKV3D-11, IGKV3D-15, IGKV3D-20, IGKV3OR2-268, IGKV4-1, IGKV5-2, IGKV6-21, IGKV6D-21, IGKV6D-41, IGLC1, IGLC2, IGLC3, IGLC6, IGLC7, IGLJ1, IGLJ2, IGLJ3, IGLJ4, IGLJ5, IGLJ6, IGLJ7, IGLV1-36, IGLV1-40, IGLV1-44, IGLV1-47, IGLV1-50, IGLV1-51, IGLV2- 8, IGLV2-11, IGLV2-14, IGLV2-18, IGLV2-23, IGLV2-33, IGLV3-1, IGLV3-9, IGLV3-10, IGLV3-12, IGLV3-16, IGLV3-19, IGLV3-21, IGLV3-22, IGLV3-25, IGLV3-27, IGLV3-32, and IGLV4-3.

13. The method of any one of claims 4-12, wherein the hybrid capture is performed using probes that are from about 30-200 nucleotides long, or at least about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more nucleotides long, or about 50-150, 50-100, 100-150, 100-120, 110-230, 120-140, or 130-150 nucleotides long.

14. The method of any one of claims 4-13, wherein the hybrid capture is performed using probes targeting from 50-500, 10-300, 50-100, 100-200, 200-300, 100-150, 150-200,200-250, or about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 different regions within BCR transcripts.

15. The method of any one of claims 4-14, wherein the hybrid capture is performed using BCR-specific probes, and wherein at least a portion of the probes target transcripts encoding an IgM, IgG, IgD, IgA, and / or IgE isotype BCR.

16. The method of any one of claims 4-15, wherein the hybrid capture is performed using BCR-specific probes, and wherein at least a portion of the probes target transcripts encoding a BCR heavy chain.

17. The method of any one of claims 4-16, wherein the hybrid capture is performed using BCR-specific probes, and wherein at least a portion of the probes target transcripts encoding a BCR light chain.

18. The method of claim 17, wherein the BCR light chains is a kappa and / or lambda light chain.

19. The method of any one of claims 4-18, wherein the hybrid capture is performed using BCR-specific probes, and wherein at least a portion of the probes target one or more constant regions of one or more BCR heavy or light chains.

20. The method of any one of claims 4-19, wherein the hybrid capture is performed using BCR-specific probes, and wherein at least a portion of the probes target one or more variable regions of one or more BCR heavy or light chains.

21. The method of any one of claims 1-20, wherein the B cells comprise transitional B cells, naive B cells, memory B cells, plasma B cells, B cell progenitors, plasmablasts, plasma cells, lymphoplasmacytoid cells, follicular B cells, marginal-zone B cells, Bl cells, peripheral B cells, and / or regulatory B cells.

22. The method of any one of claims 1-21, wherein the B cells are isolated from the bone marrow, spleen, lymph nodes, blood, lymph, secondary lymphoid organs, appendix, intestine, Peyer’s patches, tonsils, thymus, and / or lymphoid follicles.

23. The method of any one of claims 1-22, wherein the B cells comprise activated B cells.

24. The method of any one of claims 1-23, wherein the B cells comprise primary B cells.

25. The method of any one of claims 1-23, wherein the B cells are derived from a cell line.

26. The method of any one of claims 1-25, wherein the B cells express any one or more of the genes selected from CD1, CD5, CD19, CD20, CD21, CD23, CD24, CD25, CD27, CD30, CD34, CD38, CD40, CD45R, CD78, CD80, C138, CD319, Notch2, TLR4, PD-L2, IL- 6, IL-10, TGFb, CXCR3, CXCR4, CXCR5, CXCR6, IgM, IgG, IgA, IgE, and IgD.

27. The method of any one of claims 1-26, wherein during at least one of the performances of steps (z) through (zzz) each of the nucleic acid tags further comprises one or more elements selected from a capture agent, an additional sequence that allows or facilitates the detection of duplicates arising during PCR, and a NGS adapter sequence28. The method of any one of claims 4-27, wherein the hybrid capture is performed using a set of nucleic acid probes designed to capture the entire BCR repertoire present in the plurality of cells.

29. The method of any one of claims 4-28, wherein hybrid capture is performed using a set of nucleic acid probes comprising one or more probes specific for one or more of IgM, IgG, IgD, IgA, and IgE isotypes, IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2 subclasses, BCR heavy chain, BCR light chain, BCR heavy chain constant region, BCR heavy chain variable region, BCR light chain constant region, BCR light chain variable region, BCR mu heavy chain, BCR delta heavy chain, BCR gamma heavy chain, BCR epsilon heavy chain, BCR alpha heavy chain, BCR kappa light chain, BCR lambda light chain, BCR complementaritydetermining region (CDR), BCR CDR1, BCR CDR2, BCR CDR3, BCR CDR-H1, BCR CDR- H2, or BCR CDR-H3 domain.

30. The method of any one of claims 2-29, further comprising grouping sequencing reads according to one or more features selected from the group consisting of RT barcode sequences, tag barcode sequences, series or combinations of tag barcode sequences, and index sequences.

31. The method of claim 30, wherein the grouped sequencing reads are used to determine the individual B cell from among the plurality of B cells from which a given transcript originated.

32. The method of claim 30 or 31, further comprising grouping sequence reads into BCR gene sequences and non-BCR gene sequences.

33. The method of claim 32, further comprising relating the identity of the specific BCR gene sequences and / or heavy or light chain variable region sequences expressed in a given individual cell to the pattern of expression of non-BCR gene sequences in the same individual cell.

34. The method of any of claims 31-33, comprising identifying paired BCR heavy and light chain gene sequences within the same individual cell.

35. The method of any of claims 31-34, further comprising determining the relative abundance of one or more BCR clonotypes among the plurality of cells.

36. The method of any one of claims 1-35, wherein the plurality of cells were all isolated from a single subject.

37. The method of claim 36, wherein the plurality of cells comprise cells obtained at two or more different time points from the subject.

38. The method of claim 37, wherein the method comprises determining the relative abundance of one or more BCR clonotypes among the plurality of the cells, and further comprising determining the evolution of the clonotypes between the two or more different time points.

39. The method of claim 38, wherein the subject has a disease and / or has been administered a compound or other agent susceptible to affect the activity, presence, and / or role of B cells in the subject.

40. The method of any one of claims 1-39, wherein the whole transcriptome sequencing library and the BCR sequencing library are sequenced separately.

41. The method of any one of claims 1-39, wherein the whole transcriptome sequencing library and the BCR sequencing library are sequenced together.

42. The method of any one of claims 1-41, wherein one or more of steps (a), (b), (d), (e)(z), (e)(zzz), or (f) are carried out at a temperature of below about 8, 7, 6, 5, 4, 3, 2, 1, 0, -1, -2, -3, or -4 °C, between about -4 to 8, -4 to 0, 0 to 4, 4 to 8, or 0 to 8 °C, or at about 8, 7, 6, 5, 4, 3, 2, 1, 0, -1, -2, -3, or -4 °C.

43. The method of any one of claims 1-42, wherein the method comprises generating second strands of the released tagged cDNA molecules to produce double-stranded cDNA molecules prior to step (h).

44. The method of claim 43, wherein the second strands of the released cDNA molecules are generated by template switching.

45. The method of claim 44, wherein the template switching introduces a common adapter sequence to the 3 ’-end of the released cDNA molecules.

46. The method of any one of claims 1-45, wherein the nucleic acid tags are coupled to the cDNA molecules in step (e)(zz) by ligation.

47. The method of any one of claims 1-46, wherein the RT primers each comprise a 5’ overhang comprising a 5’ overhang sequence.

48. The method of any one of claims 1-47, wherein the nucleic acid tags each comprise a first strand comprising a 3’ hybridization sequence and / or a 5’ hybridization sequence flanking the 3’ end and / or the 5’ end of the tag barcode sequence, respectively.

49. The method of claim 48, wherein the RT primers each comprise a 5’ overhang comprising a 5’ overhang sequence, and wherein the nucleic acid tags each further comprise a second strand comprising: a first portion complementary to a 5’ hybridization sequence of a previously coupled nucleic acid tag or a 5’ overhang sequence of an RT primer; and a second portion complementary to the 3’ hybridization sequence.

50. The method of any one of claims 1-49, wherein one or more of the pluralities of aliquots or sublibraries are distributed in a 96-well plate.

51. The method of claim 50, wherein one or more of the 96-well plates comprise unique dual indexes.

52. The method of any one of claims 27-51, wherein the additional sequence that allows or facilitates the detection of duplicates arising during PCR comprises random or degenerate bases.

53. The method of any one of claims 27-52, wherein the capture agent comprises biotin.

54. A kit for performing any of the methods of claim 1-53.

55. The kit of claim 54, wherein the kit comprises one or more probe comprising at least about 70%, 75%, 80%, 85%, 90%, 96%, 97%, 98%, 99%, or 100% identity orcomplementarity to any one or more of the sequences shown in Table 13 or Table 14 or shown as SEQ ID NOS: 750-944, or a subsequence thereof.

56. The kit of claim 55, wherein the subsequence comprises at least about 30, 40, 50, 60, 70, 80, 90, 100, or more nucleotides.

57. A method of preparing cells or nuclei for single-cell sequencing, the method comprising:(a) providing a plurality of cells or nuclei distributed in one or more wells or containers, wherein the plurality of cells or nuclei in the one or more wells or containers are suspended in a prefixation buffer;(b) fixing the plurality of cells or nuclei by adding a cell or nucleus fixation solution to the one or more wells or containers comprising the plurality of cells or nuclei;(c) permeabilizing the plurality of cells or nuclei by adding a cell or nucleus permeabilization solution to the one or more wells or containers comprising the plurality of cells or nuclei;(d) adding a neutralization buffer to the one or more wells or containers comprising the plurality of cells or nuclei, such that the neutralization buffer prevents further fixation and / or permeabilization of the cells or nuclei;(e) freezing the plurality of cells or nuclei;(f) thawing the plurality of cells or nuclei;(g) adding cell binding beads to the plurality of thawed cells or nuclei such that the cell binding beads bind to the plurality of thawed cells or nuclei;(h) placing the one or more wells or containers on a magnet, such that the cells or nuclei bound to the cell binding beads localize to a location within each of the wells or containers in proximity to the magnet;(i) removing the supernatant from the one or more wells or containers comprising the localized plurality of cells or nuclei bound to the cell binding beads; and(j) removing the one or more wells or containers from the magnet and resuspending the plurality of cells or nuclei in the one or more wells or containers in a storage buffer.

58. The method of claim 57, wherein the supernatant is removed from the one or more wells or containers by pipetting.

59. The method of claim 57 or 58, wherein the cell binding beads are coated with concanavalin A (ConA).

60. The method of any one of claims 57-59, further comprising counting the plurality of cells or nuclei in one or more of the wells or containers prior to step (b) and subsequent to step (j), wherein the number of cells or nuclei counted subsequent to step (j) is at least 70%, 75%, 80%, 85%, 90%, or 95% of the number of cells counted prior to step (b).

61. The method of any one of claims 57-60, wherein the beads are added at a ratio of beads to cells of 4 mL beads / 10,000 cells, 6 mL beads / 50,000 cells, 9 mL beads / 75,000 cells, or 12 mL beads / 90,000 cells.

62. The method of any one of claims 57-61, wherein the cells or nuclei are any one of more of mammalian cells or nuclei, human cells or nuclei, mouse cells or nuclei, primary cells, cell lines, PBMCs, cells isolated from a tissue, HEK cells, 3T3 cells, T cells, B cells, or nuclei isolated from primary cells, cell lines, PBMCs, cells isolated from a tissue, T cells, B cells, HEK cells, 3T3 cells, or fungal cells.

63. The method of any one of claims 57-62, wherein subsequent to step (j) the cells or nuclei are used in a single cell sequencing method comprising sequencing RNA and / or genomic DNA.

64. The method of any one of claims 57-63, wherein the plurality of cells is distributed in a multi-well plate.

65. The method of claim 64, wherein the multi -well plate is a 96-well plate.

66. A method of preparing cells or nuclei for single-cell sequencing, the method comprising:(a) providing a plurality of cells or nuclei distributed in one or more wells or containers, wherein the plurality of cells or nuclei in the one or more wells or containers are suspended in a prefixation buffer;(b) fixing and permeabilizing the plurality of cells or nuclei by adding a cell or nucleus fixation solution and a permeabilization solution to the one or more wells or containers comprising the plurality of cells or nuclei;(c) adding a neutralization buffer to the one or more wells or containers comprising the plurality of cells or nuclei, such that the neutralization buffer prevents further fixation of the cells or nuclei;(d) freezing the plurality of cells or nuclei;(e) thawing the plurality of cells or nuclei;(f) adding a plurality of magnetic beads to the one or more wells or containers comprising the plurality of thawed cells or nuclei, wherein the plurality of magnetic beads are coated with an agent having affinity to molecules on the surface of the cells or nuclei such that the magnetic beads bind to the surface of the plurality of thawed cells or nuclei;(g) contacting the one or more wells or containers with a magnet such that the cells or nuclei bound to the magnetic beads become localized adjacent to the site of the magnet on the outside of each well or container;(h) removing the supernatant from the one or more wells or containers comprising the thawed plurality of cells or nuclei; and(i) resuspending the thawed plurality of cells or nuclei in the one or more wells or containers in a storage buffer.

67. The method of claim 66, further comprising performing a single-cell RNA sequencing method on the resuspended cells or nuclei.

68. The method of claim 36 or 37, further comprising performing a single-cell genomic DNA sequencing method on the resuspended cells or nuclei.

69. The method of any of claims 66-68, wherein the beads are ConA beads.

70. The method of any one of claims 66-69, wherein the plurality of cells or nuclei comprises from about 10,000 to about 100,000 cells or nuclei.

71. The method of any one of claims 66-70, wherein from about 4 mL to about 12 mL of beads are added to the plurality of cells or nuclei.

72. The method of claim 71, wherein the plurality of cells comprises from about 10,000 cells or nuclei to about 49,999 cells or nuclei, and wherein 4 mL of beads are added.

73. The method of claim 71, wherein the plurality of cells comprises from about 50,000 cells or nuclei to about 74,999 cells or nuclei, and wherein 6 mL of beads are added.

74. The method of claim 71, wherein the plurality of cells comprises from about 75,000 cells or nuclei to about 89,999 cells or nuclei, and wherein about 9 mL of beads are added.

75. The method of claim 71, wherein the plurality of cells comprises from about 90,000 to 100,000 cells or nuclei, and wherein about 12 mL of beads are added.

76. The method of any one of claims 36-44, wherein the average cell retention rate during the method is at least about 75%, 80%, 85% 90%, or 95%.

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