CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the priority benefit of U.S. Provisional Patent Application No 63/358,933, filed July 7, 2022, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under project number ZI SC 010355 by the National Institutes of Health, National Cancer Institute. The Government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 115,377 Byte Extensible Markup Language (xml) file named “767580_ST26.xml” created on July 7, 2023.

BACKGROUND

[0004] Currently, single-cell omics technologies necessarily destroy the single cells after completion of a given omics experiment. This limits the effective use of patient samples and other precious cells that are difficult to reacquire. Recently, the development of the “reusable single cell” has made it possible to reanalyze the same single cell. However, when multiple reusable single cells are subjected to multi-omics analyses, it is currently impossible to link the individual single cells with each of the omics datasets. This limitation prevents the comprehensive characterization of individual cells through multi-omics analyses. Currently, there has been no experimental method to iteratively analyze a large number of the same single cells. [0005] There is a need to identify the source cell of each single-cell omics dataset using physical evidence rather than informatic assumption.

BRIEF SUMMARY

[0006] In aspects, the disclosure provides a method of linking to a cell of origin two or more omics datasets generated from an individual reusable single cell or individual reusable single nucleus, the method comprising: (a) embedding a single cell or a single nucleus into a polyacrylamide bead; (b) covalently attaching a barcode scaffold nucleic acid to the polyacrylamide of the polyacrylamide bead, wherein the barcode scaffold nucleic acid is for constructing or attaching a unique cell barcode on the polyacrylamide bead; (c) performing a first omics experiment on the cell or nucleus, wherein the first omics experiment produces nucleic acid products; (d) attaching to the barcode scaffold nucleic acid a first cell barcode nucleic acid, wherein the sequence of the first cell barcode nucleic acid is to store and provide an unchanged reference cell identification on the barcode scaffold nucleic acid over iterative single cell omics experiments, wherein the sequence of the first cell barcode nucleic acid is also for identifying the cell of origin of omics products in the first omics experiment and link the omics products to the cell of origin; (e) sequencing the nucleic acid products produced from the first omics experiment; (f) attaching to the first cell barcode nucleic acid a second cell barcode nucleic acid, wherein the sequence of the second cell barcode nucleic acid is for linking the sequence of the second cell barcode nucleic acid and products of the second omics experiment to the first cell barcode nucleic acid after sequencing and linking the omics products to the cell of origin; (g) performing the second omics experiment on the cell or nucleus, wherein the second omics experiment produces nucleic acid products; (h) sequencing the nucleic acid products produced from the second omics experiment; (i) generating nucleic acid copies of the first and second cell barcode nucleic acids using a polymerase; (j) releasing the nucleic acid copies and sequencing the nucleic acid copies; (k) generating a reference table listing the first and second cell barcode nucleic acids; and (1) generating a combined dataset from the first and second omics experiments using the reference table; wherein the sequences of the barcode scaffold nucleic acid, first cell barcode nucleic acid, and second cell barcode nucleic acid are different from one another.

[0007] Additional aspects are as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 presents an aspect of a device of the disclosure, where the device exemplarily comprises three components.

[0009] Figures 2A-2F present schematics showing a device of the disclosure. The device allows synthesizing a cell barcode on omics DNA products and simultaneously recording the same barcode on a nucleic acid attached to a polyacrylamide bead associated with a single cell, the nucleic acid listed in the figure as the cell-barcodes recorder. Figure 2A shows a first cell barcode synthesized on both omics DNA products (proteome and transcriptome analysis) as well as the cell-barcodes recorder. Figure 2B shows a sequencing library for the proteome and transcriptome with the first cell barcode. The library can be analyzed by a high throughput sequence to acquire the sequences. Figure 2C shows a second cell barcode added to both omics DNA products (epigenome analysis, etc.) as well as the cell-barcodes recorder containing the first cell barcode. The same single cell is analyzed here with different omics methods, since the cell is re-usable within the polyacrylamide bead. Figure 2D shows a sequencing library for epigenome analysis containing the second cell barcode. The library can be analyzed by a high throughput sequencer. Figure 2E shows the device after the first and second experiments. The cell-barcodes recorder contains the first and second barcodes synthesized in the first and second experiments (Figures 2A & 2C). Figure 2F shows a sequencing library of the recorded cell barcodes. The sequencing library can be constructed from the “readout strand” of the cellbarcodes recorder using a primer and a DNA polymerase, which has strand displacement activity. Double-stranded DNA of the cell-barcode recorder can be eluted from the device. The double-stranded DNAs are used for constructing a sequencing library. One double-stranded DNA in the library contains the first and second barcodes synthesized in the first and second omics experiments. The library can be analyzed by a high throughput sequencer. The sequencing data provides a list of the first and second barcodes synthesized. The information can be used for integrating multi-omics data into individual data containers, which represent individual single cells.

[0010] Figures 3A-3C depict cell-barcode exchange from a second barcode to a third barcode. Figure 3A depicts the cell-barcodes recorder after a first omics experiment and a second omics experiment. The cell-barcodes recorder contains first and second cell barcodes. Figure 3B depicts removal of the second cell barcode. The second cell barcode can be removed by enzymatic cleavage using, e g., a Type IIS restriction enzyme. After removal, an oligo, labeled as “Addition Operator” in the figure, can be annealed and ligated to a 3’ sticky end. The first cell barcode is preserved on the device. The preserved first cell barcode can be used in data analysis as a common identifier of the same single cell. Figure 3C shows that after ligating the Addition Operator oligo, the cell-barcodes recorder is ready to record a third cell barcode in an additional omics experiment.

[0011] Figures 4A and 4B depict exemplary steps A-I that can be performed on a device as described herein (steps A-H in Figure 4A and steps I-J in Figure 4B). The figures show a cell and barcode tracking system for single-cell multi-omics analyses. Step A: A single cell can be provided with a recording function for cell barcodes. A DNA oligo for barcode recording can be immobilized onto a polyacrylamide bead. Step B: A unique cell barcode can be assigned to a single cell during single-cell RNA-seq. A unique cell barcode can be synthesized and assigned to individual single cells by DNA ligation. Step C: The DNA oligo immobilized onto the polyacrylamide bead provides a tool for recording the cell barcodes assigned to the cell. Steps D-E: The recorded cell barcode can be carried with the single cell and record all additional cell barcodes in subsequent single-cell omics experiments. The recorded cell barcode can be extracted in steps I-K and used for data analysis, for example, as in Figure 5. Steps F-G: The second cell barcode can be removed after reading out the first and second cell barcodes. Step H: A scaffold sequence “Addition Operator” oligo can be added to the first cell barcode. Subsequently, a return to step C can be made to make a record of the cell barcode for a third single-cell omics experiment. This cycle can be repeated. The first cell barcode can be preserved as a common cell identifier over all the single-cell omics experiments. Step I: An antisense chain of the recorded cell barcodes can be extracted using a primer and a DNA polymerase, which has strand displacement activity. Step J: An immobilized sense chain of the recorded cell barcodes can be retained on the device. A dissociated antisense chain can be collected from supernatant. Step K: A collected antisense chain of a recorded cell barcodes can be amplified by PCR and sequenced. The sequence data can provide a list of all cell barcodes assigned to a single cell during iterative single-cell multi-omics experiments. The steps may be changed and performed in any suitable order for cell barcode recording and are not necessarily limited to the order of the steps as shown here.

[0012] Figures 5A-5C depict a data analysis pipeline using a list of cell barcodes assigned to individual single cells. Figure 5A depicts sequenced reads from single-cell multi-omics analyses (series of single-cell omics experiments as shown in Figure 4). Boxes with solid and dashed lines indicate sequenced reads from a single cell X and Y (cells shown in Figure 5B), respectively. Each sequence has a cell barcode sequence + mRNA (cDNA), protein identifier (antibody DNA probe from, e.g., TotalSeq, BioLegend/PerkinElmer) or identifier of epigenetic marks (e.g., REpi-seq, Ohnuki et al., Genome Res., 10: 1819-1830 (2021), incorporated herein by reference). Sequencing data, e.g., from Figure 2F and Figure 4B, step K can provide lists of cell barcodes assigned to individual single cells. Figure 5C shows that the acquired lists can be used to sort the sequence reads into computerized data bins, each container representing individual single cells. The data containers can be used for single-cell multi-omics data analyses. [0013] Figures 6A-6G present selective depletion of omics DNA products derived from cells of no interest. Figure 6A presents a detailed structure of a synthesized cell barcode. Each cell barcode comprised combinations of the first, second, third, and fourth subcodes. Figure 6B shows sequences of the fourth subcodes used. Ninety-six fourth subcodes were used in the synthesis of cell barcodes. Figure 6C depicts annealing complementary oligos for the fourth subcodes. The “left” oligo and “right” oligo are also annealed to the common sequence of the synthesized cell barcode. Figure 6D depicts that annealing and ligation at 25 °C can selectively ligate the “left”, complementary and “right” oligos on the perfectly matched target. Figure 6E depicts pull-down with streptavidin beads at 37 °C. Non-ligated “left,” complementary, and “right” oligos are dissociated from the cell barcode at the temperature. Ligated “left,” complementary and “right” oligos can be retained on the target cell barcode at the temperature. Figure 6F presents optimal temperature for annealing and ligation. Melting temperatures were calculated from perfect matches (light bar, between a fourth subcode and the perfectly matched complementary oligo) and mismatch (dark bar indicates the highest melting temperature among all possible sequences of complementary oligos and each fourth subcode sequence). Figure 6G presents experimental results. Unwanted cell barcodes were depleted using streptavidin beads. Unbound DNA concentration was measured using the PicoGreen DNA quantification kit (Thermo Fisher). 100% is the 100% input before depletion with the streptavidin beads. [0014] Figure 7 presents photomicrographs that show endogenous RNases degrade most cellular RNA in a few hours.

[0015] Figure 8 presents photomicrographs that show paraformaldehyde (4%) fixation efficiently retains cellular RNA after membrane permeabilization with 0.1% NP-40.

[0016] Figure 9 presents a line graph and photomicrographs showing Acryloyl-X improves availability of RNA bases by the RNA dye of StrandBrite.

[0100] Figures 10A and 10 B show heating “re-usable single cells” at 70°C for 30 min in Tris-Acetate EDTA buffer, pH 8.5 improves availability of the RNA bases by in-cell reverse transcription. Figure 10A presents graphs showing results of extended and unextended primers collected from the re-usable single cells by treating with the RNA:DNA hybrid-specific RNase, RNase H. Products were visualized by agarose gel electrophoresis (2%) stained with SYBR Gold. Figure 10B presents a gel showing a constructed single-cell RNA-seq library from Acryloyl-X mediated re-usable single cells. Reverse transcription-dependent enrichment around 1.5-3.0 kbp was observed. The enriched smear band suggests long cDNA generation from the re-usable single cells with RNA retrieval.

[0017] Figures 11 A-l IF show automatic generation of millions of devices, as described herein. Figure 11 A presents a schematic representation of cells that have amino groups modified with monomer acrylamide. Figure 1 IB lists an acrylamide solution containing the “barcode recorder” DNA oligo, which contains 6% acrylamide/bis-acrylamide and 0.5% ammonium persulfate in TBS buffer. The “barcode recorder” DNA oligos have an aciydite modification at 5’ end, which allows their incorporation into a polyacrylamide polymer once acrylamide is polymerized, resulting in immobilization of the DNA oligo. The immobilized DNA oligo confers a recording function to the single cell. Figure 11C is a photo of a microfluidic chip that generates droplets containing the modified single cell and the acrylamide solution (average 75 pm diameter). Figure 1 ID presents a schematic representation of the droplets. Figure 1 IE presents a schematic representation of the device with a re-usable single cell and the recording function of the multiple cell barcodes. Acrylamide on amino groups in a cell is incorporated into polyacrylamide polymer by adding tetramethylethylenediamine (TEMED, final concentration 4%). Figure 1 IF is a photo showing generated devices containing the re-usable single cells in a 1.5 ml tube. Figure 11G is a microscopic image of the generated devices containing the reusable single cells stained with the RNA dye, StrandBrite.

[0018] Figures 12A-12C show a “barcode recorder” DNA on a device records a cell barcode assigned to cDNAs of a single cell in single-cell RNA-seq. Figure 12A depicts cell barcode assignment to cDNA and recording. A unique cell barcode for each cell is synthesized and assigned to cDNA using the single-cell RNA-seq method, SPLiT-seq (Rosenberg et al., Science, 360: 176-182 (2018), incorporated herein by reference). The recorded cell barcode is carried with the re-usable single cell and available for additional recording of a second single-cell omics experiment. Figure 12B is a gel showing the expected size of the recorded cell barcodes used in this experiment (arrow). Cell barcodes were copied using primers for cell barcodes and the DNA polymerase, Bst3.0. The copied cell barcodes were visualized by agarose gel electrophoresis (2%) stained with SYBR Gold. Figure 12C depicts a constructed library of single-cell RNA-seq from single cells sequenced by the Illumina sequencer, MiSeq. The synthesized cell barcode onto cDNA was confirmed by sequencing.

[0019] Figure 13 shows a reference table showing the detection of 1st and 2nd cell barcodes assigned to individual reusable single cells. Each column has a unique 1st cell barcode, and each row indicates a unique 2nd cell barcode. The enlarged portion of the reference table shows representative barcodes. The darkened color in the reference table indicates that a 1st cell barcode and a 2nd cell barcode were detected within one DNA molecule.

[0020] Figures 14A-14D show an overview of the sequencing results of the TotalSeq library generated from barcoded single cells reacted with a panel of barcoded antibodies. Figure 14A shows two representative barcodes for cell surface markers, the relative size of the barcodes in a polyacrylamide gel, and a pie graph showing the breakdown of all 666 million sequenced reads into TotalSeq products (153 million), PhiX-derived reads (261 million), and Recorded 2 cell barcodes and scMtio-seq products (252 million). Figure 14B is a graph showing the quality scores of all Readl in the TotalSeq library with the y axis showing the error probability and the x axis showing the positions of the components in Readl. Figure 14C is a graph that shows the average number of antibody molecules per antibody type (antibodies or isotype controls) per cell in young and aged mouse bone marrow cells. Figure 14D is a graph that shows the total number of antibody molecules per cell in young and aged mouse bone marrow cells.

[0021] Figure 15 shows the conventional classification of hematopoietic stem and progenitor cells by cell surface markers.

[0022] Figures 16A-16F show scatter plots for antibody evaluation used to identify different classification groups of hematopoietic stem and progenitor cells. Figure 16A is a representative scatter plot with annotations describing how to interpret a scatter plot. Figure 16B shows scatter plots of typical lineage markers such as CD3, Ly-6C, Ly-6G, CD1 lb, CD45RZB220, and TER- 119. Figure 16C shows scatter plots alternative lineage markers such as CD3, Ly-6C, Ly-6G, CD88 (C5aR) (alternative for CD1 lb), and CD20 (alternative for CD45R/B220), and TER-119. Figure 16D shows a representative scatter plot used to identify multipotent hematopoietic stem/progenitor cells and committed progenitor cells using cKit and Seal antibodies. Figure 16E shows a representative scatter plot used to identify long-term (LT) and intermediate-term (IT) hematopoietic stem cells (HSCs) using CD150 and CD43 antibodies. Figure 16F shows a representative scatter plot used to identify short-term (ST) HSCs, multipotent progenitors (MPPs), and lymphoid multipotent progenitors (LMPPs) using CD48, CD27, and CD 127 antibodies.

[0023] Figure 17 is a graph showing the number of hematopoietic stem/progenitor cells of different classification groups in a young and an aged mouse femur.

DETAILED DESCRIPTION

[0024] How individual cells respond to external changes is determined by the genes they express and their epigenome. The epigenome comprises many epigenetic markers, DNA modifications, histone modifications, and molecules that interact with the genome. Many epigenetic marks can be acquired from many individual cells as a pool of information in multiple experiments. Current cell-barcoding technologies assign unique cell barcodes to the epigenetic marks. Currently, there is a limit to the number of epigenetic markers that can be read from a single cell in a single experiment. To identify and integrate different cell barcodes assigned to one single cell, a technology is needed. Surprisingly and unexpectedly, it has been found that devices and methods disclosed herein make it possible to record and read out different cell barcodes assigned to a single cell, or nucleus, in multiple omics experiments.

[0025] In aspects, the disclosure provides a device for recording one or more barcode nucleic acids for a single cell or a single nucleus, the device comprising: (a) a polyacrylamide bead; (b) a single cell or single nucleus within the polyacrylamide bead; and (c) a nucleic acid attached to the polyacrylamide bead or the single cell or single nucleus, wherein the nucleic acid can attach to one or more barcode nucleic acids.

[0026] The term “device” as used herein is any compilation that includes the three components (a) - (c) as listed in the paragraph above.

[0027] The polyacrylamide bead, as used throughout the disclosure, can be any shape and any size that can accommodate the single nucleus or single cell. As a non-limiting example, the device overall can be spherical in shape, e.g., a bead, where the single cell or single nucleus is encompassed within the polyacrylamide bead, e.g., such that the single cell or nucleus is enveloped by or encapsulated within the polyacrylamide bead. In other aspects, the polyacrylamide bead extends inside the single cell or single nucleus, wherein the polyacrylamide bead outside the single cell or single nucleus is coextensive with the polyacrylamide bead inside the single cell or single nucleus. The single cell or nucleus can be a re-usable cell or nucleus as described in US 2020/0102604 (US 2020/0102604 is incorporated by reference herein in its entirety). In aspects of such an arrangement, the molecules comprising the scaffold outside the single cell or single nucleus can be attached to molecules comprising the scaffold inside the single cell or single nucleus, e.g., such that the scaffold penetrates into or through the cellular or nuclear membrane.

[0028] The single cell, as used throughout the disclosure, can be of any cell type, and can nucleated or non-nucleated. In aspects, the cell can be a nucleated cell type that has been enucleated, i.e., its nucleus having been removed. In aspects, the cell is a prokaryotic cell (such as a bacterial cell or an algal cell) or a eukaryotic cell (such as an insect cell, plant cell, animal cell, protozoan cell, or fungal cell). In aspects, the cell is an animal cell, e.g., a mammalian cell. In aspects, the cell is a human cell. The nucleus, as used throughout the disclosure, can be the nucleus of any nucleated cell. In aspects, the nucleus can be separated from its cell. In aspects, the nucleus is of any type of nucleated cell listed within this paragraph. [0029] The attached nucleic acid and the barcode nucleic acids as described throughout the disclosure can be of any sequence of nucleotides and can be of any number of nucleotides. The sequence of the nucleic acid can be comprised of any of the naturally occurring nucleotides (adenine (A), thymine (T), guanine (G), or cytosine (C)) or any non-natural or synthetic nucleotide suitable for replication and amplification, e.g., using polymerase chain reaction methods known in the art. In aspects, the nucleic acid is attached to the polyacrylamide bead. In aspects, the nucleic acid is attached to the single cell or single nucleus. In aspects, the nucleic acid is attached through an antibody with a conjugated nucleic acid. In aspects, the nucleic acid is covalently attached. In aspects, the nucleic acid is covalently attached through a spacer molecule.

[0030] The polyacrylamide bead, as used throughout the disclosure, is comprised of any suitable molecules, e.g., hydrophilic polymers, including those that swell in water. For example, in aspects the polyacrylamide bead can comprise polyacrylamide, poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), or polypropylene fumarate) (PPF), and the scaffold can be formed from monomers suitable for polymerizing into these polymers. The scaffold can be formed from, e.g., acrylamide, bis-acrylamide, ethylene glycol, poly erhylene glycol, vinil alcohol, poly vinyl alcohol, ethylene amine, poly erhylene amine, acrylic acid, poly acrylic acid, sodium 4- styrenesulfonate, poly 4-styrenesulfonate, allylamine hydrochloride, poly allylamine hydrochloride agarose, collagen, gelatin, etc.

[0031] In aspects, and as used throughout the disclosure, the single cell or single nucleus can be covalently attached to the polyacrylamide bead. As a non-limiting example, free amino groups of cellular components can form bonds with the polyacrylamide bead. In aspects, the single cell or single nucleus is covalently attached to the polyacrylamide bead through a spacer molecule. In aspects, the spacer molecule is formed from formaldehyde, paraformaldehyde, acrydite, an acrylamide-modified nucleotide, streptavidin, biotin, acrydite-traethyleneglycol, acrydite-polyethyleneglycol, aldehyde-modified traethyleneglycol, aldehyde-modified polyethyleneglycol, glutaraldehyde, carbodiimide, N-hydroxy succinimide ester, N-hydroxy succinimide ester modified polymer, N-hydroxy succinimide ester modified polyethyleneglycol, N-hydroxy succinimide ester modified tetra ethylene glycol, N-hydroxy succinimide ester modified polyacrylamide, immidoester, maleimide, haloacetyl, or pyridyldisufide. [0032] In aspects, the polyacrylamide bead, as used throughout the disclosure, is prepared by a) suspending a cell or nucleus in a solution comprising acrylamide and paraformaldehyde (PF A) to produce a first suspension of the cell or nucleus; b) resuspending the cell or nucleus in a solution comprising acrylamidebisacrylamide or acrylamide-N,N’-bis(acryloyl)cystamine to produce a second suspension of the cell or nucleus; and c) adding N,N,N’,N’- tetramethylethylenediamine (TEMED) to the second suspension of the cell or nucleus to polymerize the acrylamide; wherein a polyacrylamide polyacrylamide bead is formed around the single cell or nucleus.

[0033] In aspects the cell or nucleus, as used throughout the disclosure, is suspended in a solution comprising 1% to 40% acrylamide and 0. 1% to 6% PFA. In aspects, the cell or nucleus is suspended in a solution comprising 5% acrylamide to 30 % acrylamide, 10% acrylamide to 40 % acrylamide, 20% acrylamide to 30 % acrylamide, 10% acrylamide to 20 % acrylamide, 20% acrylamide, or 28% acrylamide. In aspects, the cell or nucleus is suspended in a solution comprising 10% to 30% acrylamide. In aspects, the cell or nucleus is suspended in a solution comprising 3% PFA to 5% PFA, or 4% PFA.

[0034] In aspects, the cell or nucleus, as used throughout the disclosure, is resuspended in a solution comprising 1% to 6% acrylamide-N,N’-bis(acryloyl)cystamine or 1% to 6% acrylamide-bisacrylamide. In aspects, the cell or nucleus is resuspended in a solution comprising 2% to 5% acrylamide-N,N'-bis(acryloyl)cystamine or 2% to 5% acrylamide-bisacrylamide. In aspects, the cell or nucleus is resuspended in a solution comprising 2% to 6% acrylamide-N,N'- bis(acryloyl)cystamine or 2% to 6% acrylamide-bisacrylamide. In aspects, the cell or nucleus is resuspended in a solution comprising 3% to 4% acrylamide-N,N'-bis(acryloyl)cystamine or 3% to 4% acrylamide-bisacrylamide. In aspects, the solution comprising acrylamide-bisacrylamide or acrylamide-N,N'-bis(acryloyl)cystamine further comprises Tris and ammonium persulfate.

[0035] In aspects, the polyacrylamide bead, as used throughout the disclosure, is degradable by at least one reducing agent. In aspects, the at least one reducing agent is dithiothreitol (DTT), 2-mercaptoethanol, 3 -mercapto- 1,2-propanol, 2-mercaptoethylamine, or tris (2- carboxyethyl)phosphine hydrochloride (TCEP-HCL). The reducing agent may be, e.g., any reducing agent which may cleave a disulfide bond (e.g., bonding a cellular protein to the scaffold). Once the disulfide bond is cleaved, an exposed thiol may be blocked by using alkylating reagents or oxidizing reagents. The alkylating reagents that may be used may be iodoacetamide, iodoacidic acid, or N-ethylmaleimide. The oxidizing reagents that may be used may be 5,5'-dithiobis(2-nitrobenzoic acid), 2,2'-dipyridyl disulfide, 2,6-dichloroindophenol, or oxidized form of glutathione. Other reducing agents that may be used to reduce the scaffold are EDTA or ascorbic acid. Being able to degrade the scaffold may, e.g., allow for recovery of genomic DNA of the cell or nucleus.

[0036] The polyacrylamide bead, as used throughout the disclosure, may have any suitable mesh size. In aspects of the disclosure, the polyacrylamide bead has an average mesh size of 10 nm to 40 nm. In aspects, the polyacrylamide bead has an average mesh size of 15 nm to 35 nm. In aspects, the polyacrylamide bead has an average mesh size of 33 nm.

[0037] The nucleic acid, as used throughout the disclosure, can be any nucleic acid, e.g., RNA, DNA, or a hybrid of RNA and DNA. In aspects, the nucleic acid is single-stranded. In aspects, the nucleic acid is double-stranded. In aspects, the nucleic acid is attached to the polyacrylamide bead or the single cell or single nucleus by a single strand.

[0038] A barcode nucleic acid (e.g., a barcode scaffold nucleic acid, a cell barcode nucleic acid), as used throughout the disclosure, can comprise any number of unique sequences that are used as barcode sequences, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more barcode sequences. A barcode sequence can comprise subcodes. A barcode nucleic acid can comprise any number of enzyme restriction sites (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more restriction sites). The restriction site may be any suitable site, e.g., for BbsI or Bsal.

[0039] Figure 1 presents an aspect of the disclosure comprising three components. Component 1 in the figure is a cell-barcodes recorder, where in this exemplary depiction, the recorder has two barcodes. In the figure, the cell-barcodes recorder is exemplarily a doublestranded DNA (dsDNA), which can have a function, e.g., to record multiple cell barcodes used in iterative single-cell multi-omics analyses with a single cell. Component 2 in the figure is a polymer scaffold, and component 3 is a cell. The polymer scaffold can maintain the cellbarcodes recorder and cell in the same location over multiple mixing and pooling processes, such as occurs during multi-omics analyses. The polymer scaffold can allow delivery of enzymes, antibodies, or other substances to the cell during experiments. Cellular components of the cell can be covalently attached to the polymer scaffold to retain the cellular components in the original cellular locations over iterative experiments.

[0040] Cellular components may include any of a variety of substances of which cells are composed, such as membranes, organelles, proteins, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA).

[0041] In aspects, the disclosure provides a method of making a device for recording one or more barcode nucleic acids for a single cell or single nucleus, the method comprising:

(a) suspending a single cell or single nucleus in a first solution comprising an acrylamide monomer and an aldehyde to produce a first suspension of the single cell or single nucleus;

(b) resuspending the single cell or single nucleus in a second solution to produce a second suspension of the single cell or single nucleus, the second solution comprising an acrylamide monomer, a bis-acrylamide, and a nucleic acid exogenous to the cell or nucleus; and (c) adding a polymerization initiator to the second suspension of the single cell or single nucleus to polymerize the acrylamide; to form a polyacrylamide bead that covalently attaches to cellular or nuclear components of the single cell or single nucleus through a spacer molecule formed from the aldehyde, wherein the polyacrylamide bead extends inside the single cell or single nucleus, wherein the polyacrylamide bead outside the single cell or single nucleus is coextensive with the polyacrylamide bead inside the single cell or single nucleus; and wherein the nucleic acid exogenous to the cell or nucleus attaches to the polyacrylamide bead or cellular or nuclear components of the single cell or single nucleus.

[0042] In aspects, the disclosure provides a method of making a device for recording one or more barcode nucleic acids for a single cell or single nucleus, the method comprising:

(a) treating a single cell or single nucleus with an aldehyde; (b) treating the aldehyde-treated single cell or single nucleus with succinimidyl ester of 6-((acryloyl)amino)hexanoic acid;

(c) suspending the single cell or single nucleus in a solution, the solution comprising an acrylamide monomer, a bis-acrylamide, and a nucleic acid exogenous to the cell or nucleus;

(d) adding a polymerization initiator to the suspension of (c) to polymerize the acrylamide; and

(e) heating the single cell or single nucleus; to form a polyacrylamide bead that covalently attaches to cellular or nuclear components of the single cell or single nucleus through a spacer molecule formed from the succinimidyl ester of 6-((acryloyl)amino)hexanoic acid, wherein the polyacrylamide bead extends inside the single cell or single nucleus, wherein the polyacrylamide bead outside the single cell or single nucleus is coextensive with the polyacrylamide bead inside the single cell or single nucleus; and wherein the nucleic acid exogenous to the cell or nucleus attaches to the polyacrylamide bead or cellular or nuclear components of the single cell or single nucleus.

[0043] In aspects, the nucleic acid exogenous to the cell or nucleus is RNA, DNA, or a hybrid of RNA and DNA. In aspects, the nucleic acid exogenous to the cell or nucleus is singlestranded. In aspects, the nucleic acid exogenous to the cell or nucleus is double-stranded. In aspects, the nucleic acid exogenous to the cell or nucleus becomes covalently attached by a single strand. In aspects, the nucleic acid exogenous to the cell or nucleus comprises any number of enzyme restriction sites (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more restriction sites). The restriction site may be any suitable site, e.g., for BbsI or Bsal.

[0044] In aspects, the disclosure provides methods for analyzing a genome, epigenome, transcriptome, or proteome of a single cell or a single nucleus, the method comprising: (a) taking a device as described herein; and (b) performing one or more experiments on the device of (a) to analyze the genome, epigenome, transcriptome, or proteome of the single cell or single nucleus of the device of (a).

[0045] In aspects, the disclosure provides methods for analyzing a genome, epigenome, transcriptome, or proteome of a single cell or a single nucleus, the method comprising: (a) making a device by a method as described herein; and (b) performing one or more experiments on the device of (a) to analyze the genome, epigenome, transcriptome, or proteome of the single cell or single nucleus of the device of (a).

[0046] In aspects, analysis by any method of the disclosure may be of a modification in the genome, epigenome, transcriptome, or proteome. The modification may be any modification in the genome, epigenome, transcriptome, or proteome, respectively, as compared to a control genome, epigenome, transcriptome, or proteome, respectively. The control genome, epigenome, transcriptome, or proteome may be the genome, epigenome, transcriptome, or proteome, respectively, of a normal, healthy, or wild-type single cell or single nucleus, as appropriate. A genomic modification may include genome editing, or genome engineering, and may include at least one of the following: a) Copy number variations, b) Insertions to the genome, c) Deletions in the genome, d) Alterations of genomic sequences, e) Location of deoxyribonucleic acid (DNA) modifications on the genome, f) Location of DNA methylation changes on the genome, g) Location of DNA hydroxy methylation changes on the genome, h) Location of histone modifications on chromosomes, i) Location of histone modifiers on chromosomes, j) Location of DNA bound proteins on chromosomes, k) Location of RNA polymerases on the genome/chromosomes, l) Location of ribonucleic acid (RNA) modifications on RNAs, and m) Bound proteins to RNA.

[0047] In aspects, the disclosure relates to methods to study epigenetic modifications. Antibodies that recognize specific epigenetic modifications may be used to bind to these epigenetic modifications on the genome. In aspects in the methods of the disclosure, a tag may be added to the antibody which recognizes at least one epigenetic modification. This may allow the use of more than one antibody per experiment, permitting the use of control antibodies to determine specificity of the antibody and allowing the analysis of the signal to noise ratio.

[0048] In aspects, the disclosure relates to methods for determining the location of an epigenetic modification in a genome of a single cell or single nucleus, comprising binding an antibody, peptibody, nanobody, or aptamer to the epigenetic modification on the genome; and sequencing a portion of the genome of the single cell or single nucleus comprising the epigenetic modification, to determine the location of the epigenetic modification on the genome. The epigenetic modification may be located in the region where the antibody, peptibody, nanobody, or aptamer bound. In aspects, the disclosure relates to a method for determining the location of epigenetic modification in a genome of a single cell or single nucleus, comprising preparing a single cell or single nucleus according to any of the methods described herein with respect to other aspects of the disclosure; binding an antibody to the epigenetic modification on the genome; and sequencing a portion of the genome of the single cell or single nucleus comprising the epigenetic modification, to determine the location of the epigenetic modification on the genome.

[0049] The modification in the genome, epigenome, transcriptome, or proteome may include any of a variety of modifications in the genome, epigenome, transcriptome, or proteome of the single cell or single nucleus. In this regard, the method may comprise acquiring multi-omics data in a single cell or single nucleus. Such modifications may include a modification in any one or more of a genomic sequence, DNA methylome, DNA hydroxy methylome, histone modification, binding of a histone modifier, DNA binding protein, and binding of RNA polymerase as compared to the genomic sequence, DNA methylome, DNA hydroxy methylome, histone modification, binding of a histone modifier, DNA binding protein, and binding of RNA polymerase, respectively, of a control. Further examples of modifications may include DNA methylation, histone acetylation, and histone methylation. The modification may be detected by using an antibody specific to the modification of interest. Some antibodies useful for these analyses are anti-5-methylcytosine antibody, anti-5-hydroxymethylcytosine, anti-histone H3K27ac, anti-histone H3K27me3, anti-histone H3K9ac antibody, anti-histone H3K9me3 antibody, anti -transcription factor antibody, anti -Med 1 antibody, anti -HP 1 antibody, antiHD AC 1 antibody, anti-P300 antibody, anti-STATl antibody, anti -RNA polymerase II antibody, or any other antibodies which may be useful in the acquisition of epigenomic and transcriptome data.

[0050] Using the methods of aspects of the disclosure, experiments may be repeated multiple times in the same single cell or single nucleus allowing for statistical analysis to validate the results from the single cell or single nucleus.

[0051] In aspects, the disclosure provides a method of linking to a cell of origin two or more omics datasets generated from an individual reusable single cell or individual reusable single nucleus, the method comprising: (a) embedding a single cell or a single nucleus into a polyacrylamide bead; (b) covalently attaching a barcode scaffold nucleic acid to the polyacrylamide of the polyacrylamide bead, wherein the barcode scaffold nucleic acid is for constructing or attaching a unique cell barcode on the polyacrylamide bead; (c) performing a first omics experiment on the cell or nucleus, wherein the first omics experiment produces nucleic acid products; (d) attaching to the barcode scaffold nucleic acid a first cell barcode nucleic acid, wherein the sequence of the first cell barcode nucleic acid is to store and provide an unchanged reference cell identification on the barcode scaffold nucleic acid over iterative single cell omics experiments, wherein the sequence of the first cell barcode nucleic acid is also for identifying the cell of origin of omics products in the first omics experiment and link the omics products to the cell of origin; (e) sequencing the nucleic acid products produced from the first omics experiment; (f) attaching to the first cell barcode nucleic acid a second cell barcode nucleic acid, wherein the sequence of the second cell barcode nucleic acid is for linking the sequence of the second cell barcode nucleic acid and products of the second omics experiment to the first cell barcode nucleic acid after sequencing and linking the omics products to the cell of origin; (g) performing the second omics experiment on the cell or nucleus, wherein the second omics experiment produces nucleic acid products; (h) sequencing the nucleic acid products produced from the second omics experiment; (i) generating nucleic acid copies of the first and second cell barcode nucleic acids using a polymerase; (j) releasing the nucleic acid copies and sequencing the nucleic acid copies; (k) generating a reference table listing the first and second cell barcode nucleic acids; and (1) generating a combined dataset from the first and second omics experiments using the reference table; wherein the sequences of the barcode scaffold nucleic acid, first cell barcode nucleic acid, and second cell barcode nucleic acid are different from one another.

[0052] The terms “link” and “linking” mean associating data of one experiment with data from another experiment and/or associating data of one or more experiments with an individual reusable single cell or individual reusable single nucleus. Linking the data can include analysis of the data and/or integration of the data across more than one experiment, e.g., any experiment as described herein, such as omics experiments.

[0053] In aspects, the method further comprises using a restriction enzyme to remove the second cell barcode nucleic acid from the nucleic acid of the barcode scaffold nucleic acid and the first cell barcode nucleic acid.

[0054] In aspects, the method further comprises: (i) attaching to the first cell barcode nucleic acid a third cell barcode nucleic acid, wherein the sequence of the third cell barcode nucleic acid is for linking the sequence of the third cell barcode nucleic acid and products of the third omics experiment to the first cell barcode nucleic acid after sequencing and linking the omics products to the cell of origin; (ii) performing the third omics experiment on the cell or nucleus, wherein the third omics experiment produces nucleic acid products; (iii) sequencing the nucleic acid products produced from the third omics experiment; (iv) generating nucleic acid copies of the first and third cell barcode nucleic acids using a polymerase; (v) releasing the nucleic acid copies and sequencing the nucleic acid copies; (vi) generating a reference table listing the first and third cell barcode nucleic acids; and (vii) generating a combined dataset from the first and third omics experiments using the reference table; wherein the sequences of the barcode scaffold nucleic acid, first cell barcode nucleic acid, and third cell barcode nucleic acid are different from one another

[0055] In aspects, the barcode scaffold nucleic acid is covalently attached to the polyacrylamide through a spacer molecule. In aspects, the single cell or nucleus is covalently attached to the polyacrylamide bead. In aspects, the single cell or nucleus is covalently attached to the polyacrylamide bead through a spacer molecule. In aspects, the spacer molecule is an antibody, peptibody, nanobody, peptide, or aptamer. In aspects, the spacer molecule is formed from formaldehyde, paraformaldehyde, acrydite, an acrylamide-modified nucleotide, streptavidin, biotin, acrydite-traethyleneglycol, acrydite-polyethyleneglycol, aldehyde-modified traethyleneglycol, aldehyde-modified polyethyleneglycol, glutaraldehyde, carbodiimide, N- hydroxy succinimide ester, N-hydroxy succinimide ester modified polymer, N-hydroxy succinimide ester modified polyethyleneglycol, N-hydroxy succinimide ester modified tetra ethylene glycol, N-hydroxy succinimide ester modified polyacrylamide, immidoester, maleimide, haloacetyl, or pyridyldisufide.

[0056] In aspects, the barcode scaffold nucleic acid is DNA.

[0057] In aspects, embedding the single cell or nucleus and the barcode scaffold nucleic acid into the polyacrylamide bead comprises: (A) suspending the single cell or nucleus in a first solution comprising an acrylamide monomer and an aldehyde to produce a first suspension of the cell or nucleus; (B) resuspending the cell or nucleus in a second solution to produce a second suspension of the cell or nucleus, the second solution comprising an acrylamide monomer, a bisacrylamide, and the barcode scaffold nucleic acid; and (C) adding a polymerization initiator to the second suspension of the cell or nucleus to polymerize the acrylamide; to form a polyacrylamide bead that covalently attaches to cellular or nuclear components of the cell or single nucleus through a spacer molecule formed from the aldehyde, wherein the polyacrylamide bead extends inside the cell or nucleus, wherein the polyacrylamide bead outside the cell or nucleus is coextensive with the polyacrylamide bead inside the cell or nucleus; and wherein the barcode scaffold nucleic acid is a nucleic acid exogenous to the cell or nucleus.

[0058] In aspects, embedding the single cell or nucleus and the barcode scaffold nucleic acid into the polyacrylamide bead comprises: (1) treating the single cell or single nucleus with an aldehyde; (2) treating the aldehyde-treated cell or nucleus with succinimidyl ester of 6- ((acryloyl)amino)hexanoic acid; (3) suspending the cell or nucleus in a solution, the solution comprising an acrylamide monomer, a bis-acrylamide, and the barcode scaffold nucleic acid;

(4) adding a polymerization initiator to the suspension of (3) to polymerize the acrylamide; and

(5) heating the cell or nucleus; to form the polyacrylamide bead that covalently attaches to cellular or nuclear components of the cell or nucleus through a spacer molecule formed from the succinimidyl ester of 6-((acryloyl)amino)hexanoic acid, wherein the polyacrylamide bead extends inside the cell or nucleus, wherein the polyacrylamide bead outside the cell or nucleus is coextensive with the polyacrylamide bead inside the cell or nucleus; and wherein the barcode scaffold is a nucleic acid exogenous to the cell or nucleus.

[0059] In aspects, the first or second omics experiment is for analyzing a genome, epigenome, transcriptome, or proteome of a single cell or a single nucleus.

[0060] In aspects, the method further comprises: (a) suspending the cell or nucleus in a first solution comprising an acrylamide monomer and an aldehyde to produce a first suspension of the cell or nucleus; (b) resuspending the cell or nucleus in a second solution to produce a second suspension of the cell or nucleus, the second solution comprising an acrylamide monomer and a bis-acrylamide; and (c) adding a polymerization initiator to the second suspension of the cell or nucleus to polymerize the acrylamide; to form a polyacrylamide bead that covalently attaches to cellular or nuclear components of the cell or nucleus through a spacer molecule formed from the aldehyde, wherein the polyacrylamide bead extends inside the cell or nucleus, and wherein the polyacrylamide bead outside the cell or nucleus is coextensive with the polyacrylamide bead inside the cell or nucleus. In aspects, the nucleic acid is treated within Tris-acetate ethylenediaminetetraacetic acid buffer and is heated.

[0061] In aspects, the disclosure provides a kit for preparing a device for recording one or more barcode nucleic acids for a single cell or a single nucleus, the kit comprising: (a) an acrylamide monomer; (b) an aldehyde; (c) a bis-acrylamide; (d) a polymerization initiator; and (e) a nucleic acid.

[0062] In any aspect, the single cell or single nucleus can be derived by manual isolation from a suspension, which suspension may be prepared by digestion of a solid tissue. In aspects, the single cell or single nucleus can be obtained by a method for automated or semi-automated sorting based on antibody selection or other chemical/biochemical cell characteristics. In aspects, the single cell or single nucleus can be obtained from a tissue by tissue microdissection or similar microscopy-aided technique

[0063] Suitable polymerization initiators include the following.

Chemical initiators:

Ammonium persulfate + tetramethylethylenediamine / ammonium persulfate + riboflabin Polymerization initiators used with light:

Riboflabin / (±)-Camphorquinone / Acetophenone / 4'-Hydroxyacetophenone / 3'- Hydroxyacetophenone / Benzophenone / 3 -Methylbenzophenone / 2 -Methylbenzophenone / 3,4-Dimethylbenzophenone / 3 -Hydroxybenzophenone / 4-Hydroxybenzophenone / 4,4'- Dihydroxybenzophenone / 4-Benzoylbenzoic Acid / 2-Benzoylbenzoic Acid / Methyl 2- Benzoylbenzoate / 3,3',4,4'-Benzophenonetetracarboxylic Dianhydride / 4- (Dimethylamino)benzophenone / 4,4'-Bis(dimethylamino)benzophenone / 4,4'- Bis(diethylamino)benzophenone / 4,4'-Dichlorobenzophenone / 4-Phenylbenzophenone / 1,4- Dibenzoylbenzene / 4-Benzoyl 4'-Methyldiphenyl Sulfide / Dibenzosuberenone / Benzil / p- Anisil / Methyl Benzoylformate / 9, 10-Phenanthrenequinone / 2 -Hydroxy -2- methylpropiophenone / 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone // 1 - Hydroxycyclohexyl Phenyl Ketone / Benzoin / Anisoin / Benzoin Methyl Ether / Benzoin Ethyl Ether / Benzoin Isopropyl Ether / Benzoin Isobutyl Ether / 2,2-Diethoxyacetophenone / 2,2- Dimethoxy-2-phenylacetophenone / 2-Methyl-4'-(methylthio)-2-morpholinopropiophenone / 2- Benzyl-2-(dimethylamino)-4'-morpholinobutyrophenone / 2-Isonitrosopropiophenone / 9, 10- Phenanthrenequinone / 2-Ethylanthraquinone / Sodium Anthraquinone-2-sulfonate Monohydrate / 2-Chlorothi oxanthone / l-Chloro-4-propoxy-9H-thioxanthen-9-one / 2- Isopropylthioxanthone / 2,4-Diethylthioxanthen-9-one / 2,7-Dimethoxy-9H-thioxanthen-9-one / 2,2'-Bis(2-chlorophenyl)-4,4',5,5'-tetraphenyl-l,2'-biimidaz ole / Diphenyl(2,4,6- trimethylbenzoyl)phosphine Oxide / Phenylbis(2,4,6-trimethylbenzoyl)phosphine Oxide / Lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate / Ferrocene.

Polymerization initiators used with heating: 2-(9-Oxoxanthen-2-yl)propionic Acid l,5,7-Triazabicyclo[4.4.0]dec-5-ene Salt / 2-(9- Oxoxanthen-2-yl)propionic Acid l,5-Diazabicyclo[4.3.0]non-5-ene Salt / 2-(9-Oxoxanthen-2- yl)propionic Acid l,8-Diazabicyclo[5.4.0]undec-7-ene Salt / Azobisisobutyronitrile / 2,2'- Azodi(2-methylbutyronitrile) / 2,2-‘Azobis (2,4 dimethylvaleronitryle) / Azobis(4-cyanovaleric acid) / Dimethyl 2, 2'-Azobis(2 -methylpropionate) / 2,2'-Azobis(2-methylpropionamidine) Dihydrochloride / 2,2'-Azobis[2-(2-imidazolin-2-yl)propane] Dihydrochloride / tert-Butyl Hydroperoxide / Cumene Hydroperoxide / Di-tert-butyl Peroxide / Dicumyl Peroxide / Benzoyl Peroxide.

[0064] The figures provide non-limiting exemplary uses of the devices as described herein. [0065] The following are certain aspects of the disclosure.

[0066] 1. A method of linking to a cell of origin two or more omics datasets generated from an individual reusable single cell or individual reusable single nucleus, the method comprising:

(a) embedding a single cell or a single nucleus into a polyacrylamide bead;

(b) covalently attaching a barcode scaffold nucleic acid to the polyacrylamide of the polyacrylamide bead, wherein the barcode scaffold nucleic acid is for constructing or attaching a unique cell barcode on the polyacrylamide bead;

(c) performing a first omics experiment on the cell or nucleus, wherein the first omics experiment produces nucleic acid products;

(d) attaching to the barcode scaffold nucleic acid a first cell barcode nucleic acid, wherein the sequence of the first cell barcode nucleic acid is to store and provide an unchanged reference cell identification on the barcode scaffold nucleic acid over iterative single cell omics experiments, wherein the sequence of the first cell barcode nucleic acid is also for identifying the cell of origin of omics products in the first omics experiment and link the omics products to the cell of origin;

(e) sequencing the nucleic acid products produced from the first omics experiment;

(f) attaching to the first cell barcode nucleic acid a second cell barcode nucleic acid, wherein the sequence of the second cell barcode nucleic acid is for linking the sequence of the second cell barcode nucleic acid and products of the second omics experiment to the first cell barcode nucleic acid after sequencing and linking the omics products to the cell of origin;

(g) performing the second omics experiment on the cell or nucleus, wherein the second omics experiment produces nucleic acid products;

(h) sequencing the nucleic acid products produced from the second omics experiment;

(i) generating nucleic acid copies of the first and second cell barcode nucleic acids using a polymerase;

(j) releasing the nucleic acid copies and sequencing the nucleic acid copies;

(k) generating a reference table listing the first and second cell barcode nucleic acids; and

(l) generating a combined dataset from the first and second omics experiments using the reference table; wherein the sequences of the barcode scaffold nucleic acid, first cell barcode nucleic acid, and second cell barcode nucleic acid are different from one another.

[0067] 2 The method of aspect 1, wherein the method further comprises using a restriction enzyme to remove the second cell barcode nucleic acid from the nucleic acid of the barcode scaffold nucleic acid and first cell barcode nucleic acid.

[0068] 3. The method of aspect 2, wherein the method further comprises:

(i) attaching to the first cell barcode nucleic acid a third cell barcode nucleic acid, wherein the sequence of the third cell barcode nucleic acid is for linking the sequence of the third cell barcode nucleic acid and products of the third omics experiment to the first cell barcode nucleic acid after sequencing and linking the omics products to the cell of origin,

(ii) performing the third omics experiment on the cell or nucleus, wherein the third omics experiment produces nucleic acid products;

(iii) sequencing the nucleic acid products produced from the third omics experiment;

(iv) generating nucleic acid copies of the first and third cell barcode nucleic acids using a polymerase;

(v) releasing the nucleic acid copies and sequencing the nucleic acid copies; (vi) generating a reference table listing the first and third cell barcode nucleic acids; and

(vii) generating a combined dataset from the first and third omics experiments using the reference table, wherein the sequences of the barcode scaffold nucleic acid, first cell barcode nucleic acid, and third cell barcode nucleic acid are different from one another.

[0069] 4. The method of any one of aspects 1-3, wherein the barcode scaffold nucleic acid is covalently attached to the polyacrylamide through a spacer molecule.

[0070] 5. The method of any one of aspects 1-4, wherein the single cell or nucleus is covalently attached to the polyacrylamide bead.

[0071] 6. The method of aspect 5, wherein the single cell or nucleus is covalently attached to the polyacrylamide bead through a spacer molecule.

[0072] 7. The method of aspect 6, wherein the spacer molecule is an antibody, peptibody, nanobody, peptide, or aptamer

[0073] 8. The method of aspect 6, wherein the spacer molecule is formed from formaldehyde, paraformaldehyde, acrydite, an acrylamide-modified nucleotide, streptavidin, biotin, acrydite-traethyleneglycol, acrydite-polyethyleneglycol, aldehyde-modified traethyleneglycol, aldehyde-modified polyethyleneglycol, glutaraldehyde, carbodiimide, N- hydroxy succinimide ester, N-hydroxy succinimide ester modified polymer, N-hydroxy succinimide ester modified polyethyleneglycol, N-hydroxy succinimide ester modified tetra ethylene glycol, N-hydroxy succinimide ester modified polyacrylamide, immidoester, maleimide, haloacetyl, or pyridyldisufide.

[0074] 9. The method of any one of aspects 1-8, wherein the barcode scaffold nucleic acid is DNA.

[0075] 10. The method of any one of aspects 1-9, wherein embedding the single cell or nucleus and the barcode scaffold nucleic acid into the polyacrylamide bead comprises:

(A) suspending the single cell or nucleus in a first solution comprising an acrylamide monomer and an aldehyde to produce a first suspension of the cell or nucleus; (B) resuspending the cell or nucleus in a second solution to produce a second suspension of the cell or nucleus, the second solution comprising an acrylamide monomer, a bisacrylamide, and the barcode scaffold nucleic acid; and

(C) adding a polymerization initiator to the second suspension of the cell or nucleus to polymerize the acrylamide; to form a polyacrylamide bead that covalently attaches to cellular or nuclear components of the cell or single nucleus through a spacer molecule formed from the aldehyde, wherein the polyacrylamide bead extends inside the cell or nucleus, wherein the polyacrylamide bead outside the cell or nucleus is coextensive with the polyacrylamide bead inside the cell or nucleus; and wherein the barcode scaffold nucleic acid is a nucleic acid exogenous to the cell or nucleus.

[0076J 11. The method of any one of aspects 1-10, wherein embedding the single cell or nucleus and the barcode scaffold nucleic acid into the polyacrylamide bead comprises:

(1) treating the single cell or single nucleus with an aldehyde;

(2) treating the aldehyde-treated cell or nucleus with succinimidyl ester of 6- ((acryloyl)amino)hexanoic acid;

(3) suspending the cell or nucleus in a solution, the solution comprising an acrylamide monomer, a bis-acrylamide, and the barcode scaffold nucleic acid;

(4) adding a polymerization initiator to the suspension of (3) to polymerize the acrylamide; and

(5) heating the cell or nucleus; to form the polyacrylamide bead that covalently attaches to cellular or nuclear components of the cell or nucleus through a spacer molecule formed from the succinimidyl ester of 6-((acryloyl)amino)hexanoic acid, wherein the polyacrylamide bead extends inside the cell or nucleus, wherein the polyacrylamide bead outside the cell or nucleus is coextensive with the polyacrylamide bead inside the cell or nucleus; and wherein the barcode scaffold is a nucleic acid exogenous to the cell or nucleus. [0077] 12. The method of any one of aspects 1-11, wherein the first or second omics experiment is for analyzing a genome, epigenome, transcriptome, or proteome of a single cell or a single nucleus.

[0078] 13. The method of aspect 12, wherein the method further comprises:

(a) suspending the cell or nucleus in a first solution comprising an acrylamide monomer and an aldehyde to produce a first suspension of the cell or nucleus;

(b) resuspending the cell or nucleus in a second solution to produce a second suspension of the cell or nucleus, the second solution comprising an acrylamide monomer and a bis-acrylamide; and

(c) adding a polymerization initiator to the second suspension of the cell or nucleus to polymerize the acrylamide; to form a polyacrylamide bead that covalently attaches to cellular or nuclear components of the cell or nucleus through a spacer molecule formed from the aldehyde, wherein the polyacrylamide bead extends inside the cell or nucleus, and wherein the polyacrylamide bead outside the cell or nucleus is coextensive with the polyacrylamide bead inside the cell or nucleus.

[0079] 14. The method of aspect 12 or 13, wherein the nucleic acid is treated within Trisacetate ethylenediaminetetraacetic acid buffer and is heated.

[0080] The following examples further illustrate aspects of the disclosure, but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1

[0081] This example demonstrates endogenous RNases degrade most cellular RNA in a few hours.

[0082] Mouse bone marrow cells were in 2% bovine serum albumin (BSA)/PBS, pH 7.4 for 0 or 2 hours on ice. Cellular RNAs were stained with membrane-permeable RNA-specific dye (StrandBrite, AAT Bioquest).

[0083] Results are in Figure 7. EXAMPLE 2

[0084] This example demonstrates paraformaldehyde (4%) fixation efficiently retains cellular RNA after membrane permeabilization with 0.1% NP-40.

[0085] Mouse bone marrow cells were fixed with or without ice-cold 100% methanol, 1% paraformaldehyde (PF A) and 4% PFA for 15 min. The cell membrane was permeabilized with 0.1 % NP-40 in the presence and absence of RNase inhibitors, SUPERaseHn™ RNase Inhibitor (for RNase A, B, C, 1 and Tl, Thermo Fisher) and Protector RNase Inhibitor (for RNase A, B and T2, Roche). RNA was stained with StrandBrite (AAT Bioquest).

[0086] Results are in Figure 8.

EXAMPLE 3

[0087] This example demonstrates Acryloyl-X improves availability of RNA bases by StrandBrite.

[0088] Acryloyl-X treatment following 4% PFA fixation improved availability of RNA bases, suggesting reduced modification on RNA bases and optimal retention of highly reactive amino groups. Mouse bone marrow cells were fixed with 4% PFA/PBS, pH 7.4 for 15 min. The cells were treated with 1 mg/ml Acrylol-X/PBS or 28% acrylamide/4% PFA/PBS (AA-PFA) overnight at 4°C. Subsequently, cells were encapsulated into droplets (average 75 pm diameter) containing 6% acrylamide/bis-acrylamide, 0.5% ammonium persulfate and PBS using a microfluidic chip. Acrylamide on amino groups and free acrylamide were incorporated into polyacrylamide polymer by adding Tetramethylethylenediamine (TEMED, final concentration 4%). The cells in polyacrylamide were heated at 70°C for 30 min in Tris-Acetate EDTA buffer, pH 8.5 (RNase-free, RNA retrieval). Cellular RNA was stained with StrandBrite (AAT Bioquest).

[0089] Results are shown in Figure 9.

EXAMPLE 4

[0090] This example demonstrates that heating a re-usable single cell as described in US 2020/0102604 at 70°C for 30 min in Tris-Acetate EDTA buffer, pH 8.5 improves availability of the RNA bases by in-cell reverse transcription. [0091] Mouse bone marrow cells were fixed with 4% PFA/PBS and treated with 1 mg/ml Acryloyl-X/PBS (Acryloyl-X mediated “re-usable single cells”) or 28% acrylamide/4% PFA/PBS (AA-PFA mediated “re-usable single cells”) overnight at 4°C. Cells were then encapsulated into droplets (average 75 pm diameter) containing 6% acrylamide/bis-acrylamide, 0.5% ammonium persulfate and PBS using a microfluidic chip. Acrylamide on amino groups and free acrylamide were incorporated into the polyacrylamide polymer by adding Tetramethyl ethylenediamine (TEMED, final concentration 4%). The cells were treated with or without RNA retrieval (heating at 70°C for 30 min in Tris-Acetate EDTA buffer, pH 8.5). Incell reverse transcription was performed with random primer or poly dT15 primer using SuperScript IV (Thermo Fisher).

[0092] Results are presented in Figures 10A and 10B.

EXAMPLE 5

[0093] This example demonstrates automatic generation of millions of re-usable single cells. [0094] Methods and results are in Figures 11 A-l IF.

EXAMPLE 6

[0095] This example demonstrates a barcode recorder DNA on a single cell records a cell barcode assigned to cDNAs of a single cell in single-cell RNA-seq.

[0096] Methods and results are in Figures 12A-12C.

EXAMPLE 7

[0097] This example demonstrates the detection of first and second cell barcodes assigned to individual reusable single cells.

[0098] First, a 1 ^st single-cell omics experiment (single-cell RNA-seq) was performed. Then, a 1 ^st cell barcode is assigned simultaneously to both the barcode recorder scaffold nucleic acid and the 1 ^st single-cell RNA-seq products. After the assignment of the 1 ^st cell barcode, the 2 ^nd single-cell omics experiment (Proteome, TotalSeq) was performed, and then the 2 ^nd cell barcode was assigned simultaneously to both the 1 ^st cell barcode on the barcode recorder and to the nucleic acid products of the TotalSeq. The two recorded cell barcodes were copied from reusable single cells (mouse bone marrow cells; approximately 22,000 single cells) using a DNA polymerase. The DNA products were sequenced using an Illumina sequencer. Two hundred unique reads of the two recorded cell barcodes were randomly extracted from sequenced reads. See Figure 13. Darkened cells in Figure 13 indicate when a first cell barcode and a second cell barcode were detected within one DNA molecule, indicating that the two recorded cell barcodes had successfully stored the information of the 1 ^st cell barcode and the second cell barcode assigned to the same reusable single cell from the two separate single-omics experiments. The correspondence table (as shown in Figure 13) can be used for linking each single cell-omics dataset (dataset 1, dataset 2, dataset 3, etc.) to the single cell from which it was derived. These results also suggest that the two recorded cell barcodes in one DNA molecule would allow the identification of the same reusable single cells during iterative single-cell multi-omics experiments.

EXAMPLE 8

[0099] This example demonstrates single-cell proteomic analysis of murine bone marrow hematopoietic cells using a TotalSeq library generated from barcoded single cells reacted with a panel of barcoded antibodies.

Bone marrow cells were collected from young and aged mouse sample groups. Samples were prepared as described in Example 4. Samples then underwent proteomic analysis as shown in Figure 14A. Quality scores of all Read! sequences in TotalSeq library were measured for each region of the barcodes (Figure 14B). Input number of cells is shown in Table 1. Detected unique cell barcodes on single-cell omics products (TotalSeq) are also indicated in Table 1.

Table 1

Detected number of unique single cell barcodes

[0100] To increase the accuracy of downstream data analysis, cell barcodes containing errors were removed by the software, “UMItools” (Tom Smith, Andreas Heger and Ian Sudbery. UMI- tools: modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy. Genome Research, 2017 March;27(3):491-499). Also, doublets were removed by the software, “Scrablef ’ (Samuel L. Wolock, Romain Lopez and Allon M. Klein. Scrublet: Computational Identification of Cell Doublets in Single-Cell Transcriptomic Data. Cell Systems. 2019 April 24; 8(4):281-291.e9.). The identified cell barcodes sequences were all expected sequences. These results indicated that the Split & Pool barcoding method applied to antibody- conjugated DNA generated the expected cell barcode sequences.

[0101] The total number of antibody molecules per cell was calculated for the young (1,957 molecules) and aged (2,025 molecules) cell sample (Figure 14C) in 46 million sequenced reads. The average number of antibody molecules per antibody type per antibody per cell was also calculated (Figure 14D). On average, the antibody counts/antibody type/cell of 16.8 and 17.4 antibody counts/antibody /cell for the young and aged mouse are higher than the respective isotype controls of 4.7 and 4.9. This difference between antibodies and isotype controls supports the conclusion that the reusable single cells are compatible with TotalSeq (CITE-seq).

EXAMPLE 9

[0102] This example demonstrates the identification of hematopoietic stem progenitor subpopulations using TotalSeq with reusable single cells.

[0103] Hematopoietic stem and progenitor cell subpopulations are conventionally classified by expression of the cell surface markers Lin, cKit, Seal, CD150, CD43, CD27, CD48, and CD 127 into long-term (LT) hematopoietic stem cells (HSCs), intermediate-term (IT) HSCs, short-term HSCs, multipotent progenitors (MPPs), lymphoid multipotent progenitors (LMPPs), oligopotent or lineage-restricted progenitor cells, and megakaryocyte or erythrocytic progenitors (Figure 15). [0104] To identify hematopoietic stem progenitor subpopulations, TotalSeq with reusable single cells was used as described in Example 8. FACS-like dot plots were used to evaluate antibodies as shown in Figure 16A. The typical lineage markers CD3, Ly-6C, Ly-6G, CD1 lb, CD45R/B220, and TERI 19 were used to classify the lineage positive and negative cells (Figure 16B.) The alternative lineage marker CD88 (C5aR) was used for CD1 lb and the alternative lineage marker CD20 was used for CD45R/B220 (Figure 16C). The lineage of cells was identified based on these markers.

[0105] Specifically, normalized counts of pairs of specific antibodies per cell were compared on a dot plot and assigned a lineage. Conventional identification of MPPs and lineage committed progenitors was performed by comparing cKit and Seal (Figure 16D). Conventional identification of LT HSCs and IT HSCs was performed by comparing CD 150 and CD43 (Figure 16E). ST HSCs, MPPs, and LMPPs were also identified as those cells that had been positive for cKit and Seal but had no CD 150 or CD43 binding. Conventional identification of ST HSCs, MPPs, and LMPPs was then performed by comparing CD27 and CD48 (Figure 16F).

[0106] The number of hematopoietic stem cells and progenitors in each subpopulation from the femur of a young and aged mouse were calculated based on these results (Figure 17).

[0107] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. [0108] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (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. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate 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 invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0109] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein.

Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.