Name: reusable single cell for iterative epigenomic analyses
Uploaded: 2022-02-11T13:30:00
Description: Watch this Scientific Journal Video about Reusable Single Cell for Iterative Epigenomic Analyses at JoVE.com

We thank Drs. David Sanchez-Martin and Christopher B. Buck for comments during the conceptualization stage of the project. We also thank the Genomics Core, Center for Cancer Research, National Cancer Institute, National Institutes of Health for help in preliminary experiments, and the Collaborative Bioinformatics Resource, CCR, NCI, NIH for advice in computational analysis. We thank Ms. Anna Word for helping with the optimization of DNA polymerases used in the method. This work utilized the computational resources of the NIHHPC Biowulf cluster (http://hpc.nih.gov). This project is supported by the Intramural Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health, the NCI Director&#39;s Innovation Award (#397172), and Federal funds from the National Cancer Institute under Contract No. HHSN261200800001E. We thank Drs. Tom Misteli, Carol Thiele, Douglas R. Lowy, and all members of Laboratory of Cellular Oncology for productive comments.&#160;

NOTE: A schematic representation of the method is shown in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63456/63456fig01v2.jpg" /> 
Figure 1: Schematic representation of the protocol workflow. Steps 7.2-13 are explained through schematic representations. Each row indicates a step in the protocol. A cellular protein colored in green is a human nucleosome gen...

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This article describes the step-by-step protocol for the recently reported single-cell multiepigenomic analysis using reusable single cells7. In the subsequent paragraphs, we discuss critical points, emphasizing potential limitations in the protocol. 
One of the critical points throughout the protocol (from steps 7.2-13) is avoiding DNase contamination. A single cell only has two copies of genomic DNA. Therefore, damaging genomic DNA critically reduces the signal number...

Single-cell technology is entering the era of single-cell multiomics, which integrates individual single-cell omics technologies1. Recently, single-cell transcriptomics has been combined with methods for detecting chromatin accessibility (scNMT-seq2 and SHARE-seq3) or histone modifications (Paired-seq4 and Paired-Tag5). More recently, single-cell transcriptomics and proteomics were integrated with chromatin accessibility (DOGMA-seq6). These methods use transposase-based tagging for detecting chromatin accessibility or histone modifications.
Transposase-based approaches cleave genomic DNA and add a DNA barcode at the end of the genomic DNA fragment. Each cleaved genomic fragment can only accept up to two DNA barcodes (= one epigenetic mark per cleavage site), and the genomic DNA at the cleavage site is lost. Therefore, cleavage-based approaches have a trade-off between the number of epigenetic marks tested and the signal density. This hampers the analysis of multiple epigenetic marks in the same single cell. A single-cell epigenomic method that does not cleave the genomic DNA was developed to overcome this issue7,8.
In addition to the cleavage-derived issue mentioned above, transposase-based approaches have other limitations. In single-cell epigenome analysis, it is critical to know the location of histones and DNA-associated proteins on the genome. In current approaches, this is accomplished by using unfixed single cells and retention of protein-DNA and protein-protein interactions. However, this generates strong bias to accessible chromatin regions, even in the analysis of histone modifications 9. The location of histones and genome-associated proteins on the genome can be preserved without crosslinking protein-DNA and protein-protein, using a polyacrylamide scaffold7,8. This approach reduces the structural bias observed in current approaches that depend upon protein-DNA and protein-protein interactions.
Transposase-based approaches can acquire signals only once from a single cell. Therefore, it is difficult to delineate the complete epigenome of a single cell due to the drop-off of the signals. Reusable single cells have been developed to overcome current limitations by allowing iterative epigenomic analysis in the same single cell.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x CutSmart buffer</td>
			<td>New England BioLabs</td>
			<td>B6004</td>
			<td>10x Digestion buffer</td>
		</tr>
		<tr>
			<td>200 proof ethanol</td>
			<td>Warner-Graham Company</td>
			<td>200 proof</td>
			<td>Ethanol</td>
		</tr>
		<tr>
			<td>5-Hydroxymethylcytosine (5-hmC) Monoclonal Antibody [HMC/4D9]</td>
			<td>Epigentek</td>
			<td>A-1018-100</td>
			<td>Anti-5hmC</td>
		</tr>
		<tr>
			<td>Acridine Orange/Propidium Iodide Stain</td>
			<td>Logos Biosystems</td>
			<td>F23001</td>
			<td>Cell counter</td>
		</tr>
		<tr>
			<td>Acrylamide solution, 40% in H2O, for molecular biology</td>
			<td>MilliporeSigma</td>
			<td>01697-500ML</td>
			<td>40% acrylamide solution</td>
		</tr>
		<tr>
			<td>All-in-One Fluorescence Microscope BZ-X710</td>
			<td>Keyence</td>
			<td>BZ-X710</td>
			<td>Scanning microscope</td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 Centrifugal Filter Unit</td>
			<td>MilliporeSigma</td>
			<td>UFC510024</td>
			<td>Ultrafiltration cassette</td>
		</tr>
		<tr>
			<td>Ammonium persulfate for molecular biology</td>
			<td>MilliporeSigma</td>
			<td>A3678-100G</td>
			<td>Ammonium persulfate powder</td>
		</tr>
		<tr>
			<td>Anhydrous DMF</td>
			<td>Vector laboratories</td>
			<td>S-4001-005</td>
			<td>Anhydrous N,N-dimethylformamide (DMF)</td>
		</tr>
		<tr>
			<td>Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8]</td>
			<td>Abcam</td>
			<td>ab5408</td>
			<td>Anti-Pol II</td>
		</tr>
		<tr>
			<td>Anti-TRAP220/MED1 (phospho T1457) antibody</td>
			<td>Abcam</td>
			<td>ab60950</td>
			<td>Anti-Med1</td>
		</tr>
		<tr>
			<td>BciVI</td>
			<td>New England BioLabs</td>
			<td>R0596L</td>
			<td>BciVI</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin solution, 20 mg/mL in H2O, low bioburden, protease-free, for molecular biology</td>
			<td>MilliporeSigma</td>
			<td>B8667-5ML</td>
			<td>20% BSA (Table 7)</td>
		</tr>
		<tr>
			<td>Bst DNA Polymerase, Large Fragment</td>
			<td>New England BioLabs</td>
			<td>M0275L</td>
			<td>Bst DNA polymerase</td>
		</tr>
		<tr>
			<td>BT10 Series 10 &#181;l Barrier Tip</td>
			<td>NEPTUNE</td>
			<td>BT10</td>
			<td>P10 low-retention tip</td>
		</tr>
		<tr>
			<td>CellCelector</td>
			<td>Automated Lab Solutions</td>
			<td>N/A</td>
			<td>Automated single cell picking robot</td>
		</tr>
		<tr>
			<td>CellCelector 4 nl nanowell plates for single cell cloning, Plate S200-100 100K, 24 well,ULA</td>
			<td>Automated Lab Solutions</td>
			<td>CC0079</td>
			<td>4 nL nanowell plate</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>MilliporeSigma</td>
			<td></td>
			<td>Chloroform</td>
		</tr>
		<tr>
			<td>Corning Costar 96-Well, Cell Culture-Treated, Flat-Bottom Microplate</td>
			<td>Corning</td>
			<td>3596</td>
			<td>Flat-bottom 96-well plates</td>
		</tr>
		<tr>
			<td>Deep Vent (exo-) DNA Polymerase</td>
			<td>New England BioLabs</td>
			<td>M0259L</td>
			<td>Exo- DNA polymerase</td>
		</tr>
		<tr>
			<td>DNA LoBind Tubes, 0.5 mL</td>
			<td>Eppendorf</td>
			<td>30108035</td>
			<td>0.5 mL DNA low-binding tube</td>
		</tr>
		<tr>
			<td>DNA Oligo, 1st random primer</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>1st random primer</td>
		</tr>
		<tr>
			<td>DNA Oligo, 2nd random primer Cell#01</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>2nd random primer</td>
		</tr>
		<tr>
			<td>DNA Oligo, 2nd random primer Cell#02</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>2nd random primer</td>
		</tr>
		<tr>
			<td>DNA Oligo, 2nd random primer Cell#03</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>2nd random primer</td>
		</tr>
		<tr>
			<td>DNA Oligo, 2nd random primer Cell#04</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>2nd random primer</td>
		</tr>
		<tr>
			<td>DNA Oligo, 2nd random primer Cell#05</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>2nd random primer</td>
		</tr>
		<tr>
			<td>DNA Oligo, 2nd random primer Cell#06</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>2nd random primer</td>
		</tr>
		<tr>
			<td>DNA Oligo, 2nd random primer Cell#07</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>2nd random primer</td>
		</tr>
		<tr>
			<td>DNA Oligo, 2nd random primer Cell#08</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>2nd random primer</td>
		</tr>
		<tr>
			<td>DNA Oligo, 2nd random primer Cell#09</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>2nd random primer</td>
		</tr>
		<tr>
			<td>DNA Oligo, 2nd random primer Cell#10</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>2nd random primer</td>
		</tr>
		<tr>
			<td>DNA Oligo, 2nd random primer Cell#11</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>2nd random primer</td>
		</tr>
		<tr>
			<td>DNA Oligo, 2nd random primer Cell#12</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>2nd random primer</td>
		</tr>
		<tr>
			<td>DNA Oligo, 2nd synthesis primer</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>2nd synthesis primer</td>
		</tr>
		<tr>
			<td>DNA Oligo, Ligation Adaptor</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>Ligation Adaptor</td>
		</tr>
		<tr>
			<td>DNA Oligo, Reverse Transcription primer</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>Reverse Transcription primer</td>
		</tr>
		<tr>
			<td>DNase I (RNase-free)</td>
			<td>New England BioLabs</td>
			<td>M0303L</td>
			<td>DNase I (RNase-free, 4 U).</td>
		</tr>
		<tr>
			<td>DNase I Reaction Buffer</td>
			<td>New England BioLabs</td>
			<td>B0303S</td>
			<td>10x DNase I buffer (NEB)</td>
		</tr>
		<tr>
			<td>dNTP Mix (10 mM each)</td>
			<td>Thermo Fisher</td>
			<td>R0192</td>
			<td>10 mM dNTPs</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, USA origin, Heat-inactivated</td>
			<td>MilliporeSigma</td>
			<td>F4135-500ML</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>HiScribe T7 High Yield RNA Synthesis Kit</td>
			<td>New England BioLabs</td>
			<td>E2040S</td>
			<td>In-vitro-transcription master mix</td>
		</tr>
		<tr>
			<td>Histone H3K27ac antibody</td>
			<td>Active motif</td>
			<td>39133</td>
			<td>Anti-H3K27ac</td>
		</tr>
		<tr>
			<td>Histone H3K27me3 antibody</td>
			<td>Active motif</td>
			<td>39155</td>
			<td>Anti-H3K27me3</td>
		</tr>
		<tr>
			<td>IgG from rabbit serum</td>
			<td>Millipore Sigma</td>
			<td>I5006-10MG</td>
			<td>Control IgG</td>
		</tr>
		<tr>
			<td>Iron oxide(II,III) magnetic nanopowder, 30 nm avg. part. size (TEM), NHS ester functionalized</td>
			<td>MilliporeSigma</td>
			<td>747467-1G</td>
			<td>NHS ester functionalized 30 nm iron oxide powder</td>
		</tr>
		<tr>
			<td>K-562</td>
			<td>American Type Culture Collection (ATCC)</td>
			<td>CCL-243</td>
			<td>cells</td>
		</tr>
		<tr>
			<td>Linear Acrylamide (5 mg/mL)</td>
			<td>Thermo Fisher</td>
			<td>AM9520</td>
			<td>Linear Acrylamide</td>
		</tr>
		<tr>
			<td>LUNA-FL Dual Fluorescence Cell Counter</td>
			<td>Logos Biosystems</td>
			<td>L20001</td>
			<td>Cell counter</td>
		</tr>
		<tr>
			<td>LUNA Cell Counting Slides, 50 Slides</td>
			<td>Logos Biosystems</td>
			<td>L12001</td>
			<td>Cell counter</td>
		</tr>
		<tr>
			<td>Mineral oil, BioReagent, for molecular biology, light oil</td>
			<td>MilliporeSigma</td>
			<td>M5904-500ML</td>
			<td>Mineral oil</td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242;-Tetramethylethylenediamine for molecular biology</td>
			<td>MilliporeSigma</td>
			<td>T7024-100ML</td>
			<td>N,N,N&#8242;,N&#8242;-Tetramethylethylenediamine</td>
		</tr>
		<tr>
			<td>NaCl (5 M), RNase-free</td>
			<td>Thermo Fisher</td>
			<td>AM9760G</td>
			<td>5M NaCl</td>
		</tr>
		<tr>
			<td>NanoDrop Lite</td>
			<td>Thermo Fisher</td>
			<td>2516</td>
			<td>&#160;Microvolume spectrophotometer</td>
		</tr>
		<tr>
			<td>NEST 2 mL 96-Well Deep Well Plate, V Bottom</td>
			<td>Opentrons</td>
			<td>N/A</td>
			<td>2 mL deep well 96-well plate</td>
		</tr>
		<tr>
			<td>Non-skirted 96-well PCR plate</td>
			<td>Genesee Scientific</td>
			<td>27-405</td>
			<td>96-well PCR plate</td>
		</tr>
		<tr>
			<td>NuSive GTG Agarose</td>
			<td>Lonza</td>
			<td>50081</td>
			<td>Agarose</td>
		</tr>
		<tr>
			<td>OmniPur Acrylamide: Bis-acrylamide 19:1, 40% Solution</td>
			<td>MilliporeSigma</td>
			<td>1300-500ML</td>
			<td>40%Acrylamide/Bis-acrylamide</td>
		</tr>
		<tr>
			<td>OT-2 lab robot</td>
			<td>Opentrons</td>
			<td>OT2</td>
			<td>Automated liquid handling robot</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, EM Grade, Purified, 20% Aqueous Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713</td>
			<td>20% Pararmaldehyde</td>
		</tr>
		<tr>
			<td>PBS (10x), pH 7.4</td>
			<td>Thermo Fisher</td>
			<td>70011044</td>
			<td>10x PBS</td>
		</tr>
		<tr>
			<td>PIPETMAN Classic P1000</td>
			<td>GILSON</td>
			<td>F123602</td>
			<td>A P1000 pipette</td>
		</tr>
		<tr>
			<td>Protein LoBind Tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>925000090</td>
			<td>1.5 mL Protein low-binding tube</td>
		</tr>
		<tr>
			<td>QIAgen Gel Extraction kit</td>
			<td>Qiagen</td>
			<td>28706</td>
			<td>A P1000 pipette</td>
		</tr>
		<tr>
			<td>Quant-iT PicoGreen dsDNA Assay</td>
			<td>Thermo Fisher</td>
			<td>P11495</td>
			<td>&#160;dsDNA specific intercalator dye</td>
		</tr>
		<tr>
			<td>Quick Ligation kit</td>
			<td>New England BioLabs</td>
			<td>M2200L</td>
			<td>T4 DNA ligase (NEB)</td>
		</tr>
		<tr>
			<td>RNaseOUT Recombinant Ribonuclease Inhibitor</td>
			<td>Thermo Fisher</td>
			<td>10777019</td>
			<td>RNAse inhibitor</td>
		</tr>
		<tr>
			<td>S-4FB Crosslinker (DMF-soluble)</td>
			<td>Vector laboratories</td>
			<td>S-1004-105</td>
			<td>Succinimidyl 4-formylbenzoate (S-4FB)</td>
		</tr>
		<tr>
			<td>S-HyNic</td>
			<td>Vector laboratories</td>
			<td>S-1002-105</td>
			<td>Succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HyNic)</td>
		</tr>
		<tr>
			<td>Sodium Acetate, 3 M, pH 5.2, Molecular Biology Grade</td>
			<td>MilliporeSigma</td>
			<td>567422-100ML</td>
			<td>3M Sodium acetate (pH 5.2)</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate, 1M buffer soln., pH 8.5</td>
			<td>Alfa Aesar</td>
			<td>J60408</td>
			<td>1M sodium bicarbonate buffer, pH 8.5</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic for molecular biology</td>
			<td>MilliporeSigma</td>
			<td>S3264-250G</td>
			<td>Na2HPO4</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic for molecular biology</td>
			<td>MilliporeSigma</td>
			<td>S3139-250G</td>
			<td>NaH2PO4</td>
		</tr>
		<tr>
			<td>SuperScript IV reverse transcriptase</td>
			<td>Thermo Fisher</td>
			<td>18090050</td>
			<td>Reverse transcriptase</td>
		</tr>
		<tr>
			<td>SYBR Gold Nucleic Acid Gel Stain (10,000x Concentrate in DMSO)</td>
			<td>Thermo Fisher</td>
			<td>S11494</td>
			<td>An intercalator dye for DNA</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase Reaction Buffer</td>
			<td>New England BioLabs</td>
			<td>B0202S</td>
			<td>10x T4 DNA ligase reaction buffer</td>
		</tr>
		<tr>
			<td>ThermoPol Reaction Buffer Pack</td>
			<td>New England BioLabs</td>
			<td>B9004S</td>
			<td>10x TPM-T buffer (Tris-HCl/Pottasium chloride/Magnesium sulfate/Triton X-100)</td>
		</tr>
		<tr>
			<td>TRIzol LS reagent</td>
			<td>Thermo Fisher</td>
			<td>10296-028</td>
			<td>Guanidinium thiocyanate-phenol-chloroform extraction</td>
		</tr>
		<tr>
			<td>TruSeq Nano DNA library prep kit</td>
			<td>Illumina</td>
			<td>20015965</td>
			<td>A DNA library preparation kit (see also the manufacturer&#39;s instruction)</td>
		</tr>
		<tr>
			<td>Ultramer DNA Oligo, Anti-5hmC_Ab#005</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>An amine-modified DNA probe for antibody</td>
		</tr>
		<tr>
			<td>Ultramer DNA Oligo, Anti-H3K27ac_Ab#002</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>An amine-modified DNA probe for antibody</td>
		</tr>
		<tr>
			<td>Ultramer DNA Oligo, Anti-H3K27me3_Ab#003</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>An amine-modified DNA probe for antibody</td>
		</tr>
		<tr>
			<td>Ultramer DNA Oligo, Anti-Med1_Ab#004</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>An amine-modified DNA probe for antibody</td>
		</tr>
		<tr>
			<td>Ultramer DNA Oligo, Anti-Pol II_Ab#006</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>An amine-modified DNA probe for antibody</td>
		</tr>
		<tr>
			<td>Ultramer DNA Oligo, Control IgG_Ab#001</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A, see Table 3</td>
			<td>An amine-modified DNA probe for control IgG</td>
		</tr>
		<tr>
			<td>UltraPure 0.5 M EDTA, pH 8.0</td>
			<td>Thermo Fisher</td>
			<td>15575020</td>
			<td>0.5M EDTA, pH 8.0</td>
		</tr>
		<tr>
			<td>UltraPure DNase/RNase-Free Distilled Water</td>
			<td>Thermo Fisher</td>
			<td>10977023</td>
			<td>Ultrapure water</td>
		</tr>
		<tr>
			<td>Zeba Splin Desalting Columns, 7K MWCO, 0.5 mL</td>
			<td>Thermo Fisher</td>
			<td>89882</td>
			<td>Desalting column</td>
		</tr>
	</tbody>
</table>

reusable single cell for iterative epigenomic analyses

Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a single cell and requiring data from other cells to distinguish signal from experimental background noise in a single cell. This paper describes a method to reuse the same single cell for iterative epigenomic analyses. 
In this experimental method, cellular proteins are first anchored to a polyacrylamide polymer instead of crosslinking them to protein and DNA, alleviating structural bias. This critical step allows repeated experiments with the same single cell. Next, a random primer with a scaffold sequence for proximity ligation is annealed to the genomic DNA, and the genomic sequence is added to the primer by extension using a DNA polymerase. Subsequently, an antibody against an epigenetic marker and control IgG, each labeled with different DNA probes, are bound to the respective targets in the same single cell. 
Proximity ligation is induced between the random primer and the antibody by adding a connector DNA with complementary sequences to the scaffold sequence of the random primer and the antibody-DNA probe. This approach integrates antibody information and nearby genome sequences in a single DNA product of proximity ligation. By enabling repeated experiments with the same single cell, this method allows an increase in data density from a rare cell and statistical analysis using only IgG and antibody data from the same cell. The reusable single cells prepared by this method can be stored for at least a few months and reused later to broaden epigenetic characterization and increase data density. This method provides flexibility to researchers and their projects.

K562 single cells were generated using the protocol described in step 8 (see Figure 5). Single cells were embedded in the outer layer of the polyacrylamide bead. Cell DNA was stained and visualized using an intercalator dye for DNA staining.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63456/63456fig05.jpg" /> 
Figure 5: Generated reusable single cells. Cells are sta...

Watch this Scientific Journal Video about Reusable Single Cell for Iterative Epigenomic Analyses at JoVE.com

Reusable Single Cell for Iterative Epigenomic Analyses

The present protocol describes a single-cell method for iterative epigenomic analyses using a reusable single cell. The reusable single cell allows analyses of multiple epigenetic marks in the same single cell and statistical validation of the results.

Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic ...

This experimental method permits the long-term storage of the reusable single cells for epigenomic testing. It also allows the analysis of many epigenetic marks and transcription factors in the same single cell. In current methods for single-cell epigenome analysis, the same single cells cannot be reanalyzed.However, with reusable single cells, the same single cells can be reanalyzed many times. This technique is useful for diagnosis and design of epigenetic therapy, particularly when patient cells are limited in number. This method is most suitable to the study of the epigenome, the regulation of gene expression, and other systems as long as specific antibodies for epigenetic marks are available.When you first try this technique, we recommend that you practice on samples that are not too precious, and you validate the experiment by using shallow sequencing such as MiSeq or iSeq 100. To begin, in a clean laminar flow hood, mix one milliliter of cell suspension and 199 milliliters of one millimolar EDTA PBS containing an intercalator dye for DNA. After mixing, transfer 200 microliters of the cell suspension to each well of the flat bottom 96-well plate.Put the cover onto the 96-well plate and scan the plate using a scanning microscope. Next, tilt the plate and wait for a few minutes until the single cell has sunk to the lower edge of the well. Then place the pipette tip on the bottom corner and transfer the single cell and the buffer to a PCR tube.Check the well using a fluorescence microscope to confirm the transfer. If a single cell is still in the well, add 200 microliters per well of one millimolar EDTA PBS containing an intercalator dye for DNA and repeat the transfer. After the transfer, place a cover on the 96-well PCR plate and centrifuge the plate using a swing rotor without a break.After centrifugation, place the plate on the deck of an automatic liquid handling robot. Next, drag and drop the supplemental code one to the software window of the liquid-handling robot and run the program to remove 195 microliters of the supernatant with a very slow pipetting speed. Add 50 microliters of 4%TEMED or mineral oil and incubate the plate overnight at room temperature.The next day, remove the mineral oil by pipetting and wash the cells five times with 200 microliters of TP magnesium negative buffer. After the last wash, remove the supernatant, add 15 microliters of annealing buffer, and incubate on ice for one hour. After incubation, place the PCR tubes on a thermal cycler and heat at 94 degrees Celsius for three minutes.Then transfer the tubes to an ice-cooled metal block and incubate for two minutes. Next, add four microliters of magnesium sulfate-sodium chloride, DNTP mix and mix by vortexing at a maximum speed. Then add one microliter of BST polymerase large fragment, mix by vortexing, and incubate for four hours on a shaker.After incubation, place the PCR tube on a thermal cycler and run a four-hour or overnight program as described in the text. For antibody binding, add 1.625 microliters of sodium chloride EDTA BSA solution to the tube. Mix by vortexing at low speed and incubate the tube on ice for one hour.Then add 0.1 micrograms per milliliter of antibody and control IgG conjugated with the antibody probe. After adding the antibodies, incubate the tubes on ice with gentle shaking. The next day, wash the cells twice with 200 microliters of TPM-T buffer on ice.Then remove the supernatant and wash once with T4 DNA ligase buffer. Next, add 19 microliters of ligation adapter solution and incubate for one hour at 25 degrees Celsius. Then, add one microliter T4 DNA ligase and mix the tube by vortexing at medium speed.Place the tube on a thermal cycler and run the proximity ligation program. After the run, wash the cells twice with 200 microliters of BST magnesium negative EDTA positive buffer and store the cells overnight at four degrees Celsius. The following day, wash the cells twice with 200 microliters of BST magnesium negative EDTA negative buffer, and add 20 microliters of a mixture containing BST, DNTPs, and magnesium sulfate.Then mix with a vortex mixer and incubate the tube for four hours on an orbital shaker. After incubation, place the tube on a thermal cycler and run the full extension program as described in the text manuscript. Next, for multiple displacement amplification, add 0.4 microliters of 100 micromolar second random primer, and mix by vortexing.Then, incubate the tube for two hours on an orbital shaker before placing the tube on a thermal cycler for heating at 94 degrees Celsius. After three minutes, place the tubes in an ice-cooled metal block and add one microliter of BST DNA polymerase. Vortex the tubes at slow speed.Then place the tubes on a thermal cycler to run the multiple displacement amplification program. After the program, transfer approximately 20 microliters of supernatant to a PCR tube, and store it at minus 80 degrees Celsius. Next, add 20 microliters of 0.05%TWEEN 20 and 0.1X TE buffer to a PCR tube containing the reusable single cell, and incubate the tube overnight at four degrees Celsius.The following day, collect the supernatant and combine it with the supernatant collected previously. Then, soak the reusable single cell and TBS buffer containing 50%glycerol, five millimolar EDTA, 0.5%BSA, and 0.05%TWEEN 20, then incubate it for 30 minutes on an orbital shaker. After incubation, store the reusable single cell at minus 20 degrees Celsius until further use.Finally, add 40 microliters of Exo negative master mix, and place the tube on a thermal cycler to run the program as described in the text manuscript. K562 reusable single cells were generated using this protocol. Single cells were embedded in the outer layer of the polyacrylamide bead and visualized using an intercalator dye for DNA staining.DNA products before and after BciVI digestion are shown here. The restriction enzyme digestion generated the expected 19 to 20, 31 to 32, 49, and greater than 49 base pair fragments. Further, the constructed DNA library was analyzed by capillary electrophoresis for the desired products.The sequencing results indicated that the unique mapped reads from anti-histone H3 acetylated lysine 27 and anti-histone H3 trimethylated lysine 27 were significantly more numerous than from the control IgG. The REpi sequencing signals were evaluated by comparing REpi sequencing data with bulk ChIP sequencing data. On average, approximately 91%of the histone H3 acetylated lysine 27 signals from REpi sequencing overlapped with histone H-3 acetylated lysine 27 peaks in bulk ChIP sequencing.The precision of REpi sequencing for the inactive histone mark, histone H3 trimethylated lysine 27 was 52%The sensitivity of REpi sequencing was analyzed to measure how many peaks of bulk ChIP sequencing were detected by REpi sequencing reproduced reads. The sensitivity of REpi sequencing was 55.30%in histone H3 acetylated lysine 27 and 50.94%in histone H3 trimethylated lysine 27. Please make sure you use DNS-free reagents.Also, all operations that involve the opening tube leads or reagent leads should be performed in a clean hood. DNA products of this procedure are sequenced and then mapped to the genome to reveal which histone modifications are present, in which regions of the genome in single cells. This technology made it possible to analyze multiple epigenetic marks in the same single cell and paved the way for multiomics analysis in any single cell.

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