Reusable Single Cell for Iterative Epigenomic Analyses

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

NOTE: A schematic representation of the method is shown in Figure 1.

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 generated based on a crystal structure (PDB: 6M4G).

Results

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.

Figure 5: Generated reusable single cells. Cells are sta.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
10x CutSmart buffer	New England BioLabs	B6004	10x Digestion buffer
200 proof ethanol	Warner-Graham Company	200 proof	Ethanol
5-Hydroxymethylcytosine (5-hmC) Monoclonal Antibody [HMC/4D9]	Epigentek	A-1018-100	Anti-5hmC
Acridine Orange/Propidium Iodide Stain	Logos Biosystems	F23001	Cell counter
Acrylamide solution, 40% in H2O, for molecular biology	MilliporeSigma	01697-500ML	40% acrylamide solution
All-in-One Fluorescence Microscope BZ-X710	Keyence	BZ-X710	Scanning microscope
Amicon Ultra-0.5 Centrifugal Filter Unit	MilliporeSigma	UFC510024	Ultrafiltration cassette
Ammonium persulfate for molecular biology	MilliporeSigma	A3678-100G	Ammonium persulfate powder
Anhydrous DMF	Vector laboratories	S-4001-005	Anhydrous N,N-dimethylformamide (DMF)
Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8]	Abcam	ab5408	Anti-Pol II
Anti-TRAP220/MED1 (phospho T1457) antibody	Abcam	ab60950	Anti-Med1
BciVI	New England BioLabs	R0596L	BciVI
Bovine Serum Albumin solution, 20 mg/mL in H2O, low bioburden, protease-free, for molecular biology	MilliporeSigma	B8667-5ML	20% BSA (Table 7)
Bst DNA Polymerase, Large Fragment	New England BioLabs	M0275L	Bst DNA polymerase
BT10 Series 10 µl Barrier Tip	NEPTUNE	BT10	P10 low-retention tip
CellCelector	Automated Lab Solutions	N/A	Automated single cell picking robot
CellCelector 4 nl nanowell plates for single cell cloning, Plate S200-100 100K, 24 well,ULA	Automated Lab Solutions	CC0079	4 nL nanowell plate
Chloroform	MilliporeSigma		Chloroform
Corning Costar 96-Well, Cell Culture-Treated, Flat-Bottom Microplate	Corning	3596	Flat-bottom 96-well plates
Deep Vent (exo-) DNA Polymerase	New England BioLabs	M0259L	Exo- DNA polymerase
DNA LoBind Tubes, 0.5 mL	Eppendorf	30108035	0.5 mL DNA low-binding tube
DNA Oligo, 1st random primer	Integrated DNA Technologies	N/A, see Table 3	1st random primer
DNA Oligo, 2nd random primer Cell#01	Integrated DNA Technologies	N/A, see Table 3	2nd random primer
DNA Oligo, 2nd random primer Cell#02	Integrated DNA Technologies	N/A, see Table 3	2nd random primer
DNA Oligo, 2nd random primer Cell#03	Integrated DNA Technologies	N/A, see Table 3	2nd random primer
DNA Oligo, 2nd random primer Cell#04	Integrated DNA Technologies	N/A, see Table 3	2nd random primer
DNA Oligo, 2nd random primer Cell#05	Integrated DNA Technologies	N/A, see Table 3	2nd random primer
DNA Oligo, 2nd random primer Cell#06	Integrated DNA Technologies	N/A, see Table 3	2nd random primer
DNA Oligo, 2nd random primer Cell#07	Integrated DNA Technologies	N/A, see Table 3	2nd random primer
DNA Oligo, 2nd random primer Cell#08	Integrated DNA Technologies	N/A, see Table 3	2nd random primer
DNA Oligo, 2nd random primer Cell#09	Integrated DNA Technologies	N/A, see Table 3	2nd random primer
DNA Oligo, 2nd random primer Cell#10	Integrated DNA Technologies	N/A, see Table 3	2nd random primer
DNA Oligo, 2nd random primer Cell#11	Integrated DNA Technologies	N/A, see Table 3	2nd random primer
DNA Oligo, 2nd random primer Cell#12	Integrated DNA Technologies	N/A, see Table 3	2nd random primer
DNA Oligo, 2nd synthesis primer	Integrated DNA Technologies	N/A, see Table 3	2nd synthesis primer
DNA Oligo, Ligation Adaptor	Integrated DNA Technologies	N/A, see Table 3	Ligation Adaptor
DNA Oligo, Reverse Transcription primer	Integrated DNA Technologies	N/A, see Table 3	Reverse Transcription primer
DNase I (RNase-free)	New England BioLabs	M0303L	DNase I (RNase-free, 4 U).
DNase I Reaction Buffer	New England BioLabs	B0303S	10x DNase I buffer (NEB)
dNTP Mix (10 mM each)	Thermo Fisher	R0192	10 mM dNTPs
Fetal Bovine Serum, USA origin, Heat-inactivated	MilliporeSigma	F4135-500ML	Fetal bovine serum
HiScribe T7 High Yield RNA Synthesis Kit	New England BioLabs	E2040S	In-vitro-transcription master mix
Histone H3K27ac antibody	Active motif	39133	Anti-H3K27ac
Histone H3K27me3 antibody	Active motif	39155	Anti-H3K27me3
IgG from rabbit serum	Millipore Sigma	I5006-10MG	Control IgG
Iron oxide(II,III) magnetic nanopowder, 30 nm avg. part. size (TEM), NHS ester functionalized	MilliporeSigma	747467-1G	NHS ester functionalized 30 nm iron oxide powder
K-562	American Type Culture Collection (ATCC)	CCL-243	cells
Linear Acrylamide (5 mg/mL)	Thermo Fisher	AM9520	Linear Acrylamide
LUNA-FL Dual Fluorescence Cell Counter	Logos Biosystems	L20001	Cell counter
LUNA Cell Counting Slides, 50 Slides	Logos Biosystems	L12001	Cell counter
Mineral oil, BioReagent, for molecular biology, light oil	MilliporeSigma	M5904-500ML	Mineral oil
N,N,N′,N′-Tetramethylethylenediamine for molecular biology	MilliporeSigma	T7024-100ML	N,N,N′,N′-Tetramethylethylenediamine
NaCl (5 M), RNase-free	Thermo Fisher	AM9760G	5M NaCl
NanoDrop Lite	Thermo Fisher	2516	Microvolume spectrophotometer
NEST 2 mL 96-Well Deep Well Plate, V Bottom	Opentrons	N/A	2 mL deep well 96-well plate
Non-skirted 96-well PCR plate	Genesee Scientific	27-405	96-well PCR plate
NuSive GTG Agarose	Lonza	50081	Agarose
OmniPur Acrylamide: Bis-acrylamide 19:1, 40% Solution	MilliporeSigma	1300-500ML	40%Acrylamide/Bis-acrylamide
OT-2 lab robot	Opentrons	OT2	Automated liquid handling robot
Paraformaldehyde, EM Grade, Purified, 20% Aqueous Solution	Electron Microscopy Sciences	15713	20% Pararmaldehyde
PBS (10x), pH 7.4	Thermo Fisher	70011044	10x PBS
PIPETMAN Classic P1000	GILSON	F123602	A P1000 pipette
Protein LoBind Tubes, 1.5 mL	Eppendorf	925000090	1.5 mL Protein low-binding tube
QIAgen Gel Extraction kit	Qiagen	28706	A P1000 pipette
Quant-iT PicoGreen dsDNA Assay	Thermo Fisher	P11495	dsDNA specific intercalator dye
Quick Ligation kit	New England BioLabs	M2200L	T4 DNA ligase (NEB)
RNaseOUT Recombinant Ribonuclease Inhibitor	Thermo Fisher	10777019	RNAse inhibitor
S-4FB Crosslinker (DMF-soluble)	Vector laboratories	S-1004-105	Succinimidyl 4-formylbenzoate (S-4FB)
S-HyNic	Vector laboratories	S-1002-105	Succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HyNic)
Sodium Acetate, 3 M, pH 5.2, Molecular Biology Grade	MilliporeSigma	567422-100ML	3M Sodium acetate (pH 5.2)
Sodium bicarbonate, 1M buffer soln., pH 8.5	Alfa Aesar	J60408	1M sodium bicarbonate buffer, pH 8.5
Sodium phosphate dibasic for molecular biology	MilliporeSigma	S3264-250G	Na2HPO4
Sodium phosphate monobasic for molecular biology	MilliporeSigma	S3139-250G	NaH2PO4
SuperScript IV reverse transcriptase	Thermo Fisher	18090050	Reverse transcriptase
SYBR Gold Nucleic Acid Gel Stain (10,000x Concentrate in DMSO)	Thermo Fisher	S11494	An intercalator dye for DNA
T4 DNA Ligase Reaction Buffer	New England BioLabs	B0202S	10x T4 DNA ligase reaction buffer
ThermoPol Reaction Buffer Pack	New England BioLabs	B9004S	10x TPM-T buffer (Tris-HCl/Pottasium chloride/Magnesium sulfate/Triton X-100)
TRIzol LS reagent	Thermo Fisher	10296-028	Guanidinium thiocyanate-phenol-chloroform extraction
TruSeq Nano DNA library prep kit	Illumina	20015965	A DNA library preparation kit (see also the manufacturer's instruction)
Ultramer DNA Oligo, Anti-5hmC_Ab#005	Integrated DNA Technologies	N/A, see Table 3	An amine-modified DNA probe for antibody
Ultramer DNA Oligo, Anti-H3K27ac_Ab#002	Integrated DNA Technologies	N/A, see Table 3	An amine-modified DNA probe for antibody
Ultramer DNA Oligo, Anti-H3K27me3_Ab#003	Integrated DNA Technologies	N/A, see Table 3	An amine-modified DNA probe for antibody
Ultramer DNA Oligo, Anti-Med1_Ab#004	Integrated DNA Technologies	N/A, see Table 3	An amine-modified DNA probe for antibody
Ultramer DNA Oligo, Anti-Pol II_Ab#006	Integrated DNA Technologies	N/A, see Table 3	An amine-modified DNA probe for antibody
Ultramer DNA Oligo, Control IgG_Ab#001	Integrated DNA Technologies	N/A, see Table 3	An amine-modified DNA probe for control IgG
UltraPure 0.5 M EDTA, pH 8.0	Thermo Fisher	15575020	0.5M EDTA, pH 8.0
UltraPure DNase/RNase-Free Distilled Water	Thermo Fisher	10977023	Ultrapure water
Zeba Splin Desalting Columns, 7K MWCO, 0.5 mL	Thermo Fisher	89882	Desalting column