Detailed protocol ─ Chromatin Integration labeling

doi:10.1038/protex.2018.122

Method Article

Detailed protocol ─ Chromatin Integration labeling

https://doi.org/10.1038/protex.2018.122

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Chromatin plays a crucial role in gene regulation, and chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been the standard technique for examining protein–DNA interactions across the whole genome. However, it is difficult to obtain epigenomic information from limited numbers of cells by ChIP-seq because of sample loss during chromatin preparation and inefficient immunoprecipitation. In this study, we established an immunoprecipitation-free epigenomic profiling method named Chromatin Integration Labeling (ChIL), which enables the amplification of genomic sequences closely associated with the target molecules, before cell lysis. Using ChIL followed by sequencing (ChIL-seq), we reliably detected the distributions of histone modifications and DNA-binding factors in 100 to 1,000 cells. In addition, ChIL-seq successfully detected genomic regions associated with histone marks at the single-cell level. ChIL-seq thus offers an alternative method to ChIP-seq for epigenomic profiling using small numbers of cells, in particular those attached to culture plates and after immunofluorescence.

Cell biology

Genetics

chromatin

epigenetics

gene regulation

histone modification

transcription factor

ChIP-seq

For identifying protein–DNA interactions across the genome, ChIP-seq has been used as the standard technique. The original ChIP-seq method required typically a million of (at least 10⁴) cells, because samples can be lost during chromatin preparation and immunoprecipitation, particularly when using crosslinked chromatin^1-4. To start with smaller numbers of cells, several low-input methods have been developed^5-19, e.g. by using improved micrococcal nuclease treatment⁷, transposase tagging¹¹, T7 RNA polymerase-based linear amplification¹⁴, adaptor ligation^{15, 16, 18}, droplet technologies¹⁰, and a special microfluidic device⁸. Recently, an immunoprecipitation-free CUT&RUN technique, based on the chromatin immunocleavage (ChIC)20, has been reported as a convenient and efficient low-input method^{17, 19, 20}. While an ultimate goal of low-input approach is to establish single-cell epigenome/protein-binding profiling, even the method thus far closest to this goal still suffers from low genome coverage¹⁰. Here we show ChIL (Chromatin Integration Labeling) technique, which combines immunostaining, transposase tagging, and linear amplification, can be useful for low-input epigenome/protein-binding profiling even from single cells. ChIL also opens a way to link immunofluorescence and epigenome information in the same cells.

Cell lines of interest
DMEM (Nacalai Tesque, 08458-16)
L-Glutamine–Penicillin–Streptomycin solution (Sigma Aldrich, G6784)
FBS (Gibco, 10270-106)
2.5g/l-Trypsin/1mmol/l-EDTA solution (Nacalai Tesque, 3555464)
10x D-PBS(-) (Wako, 048-29805)
16% Paraformaldehyde (Electron Microscopy Sciences, 15710-S)
Polyethylene Glycol Mono-p-isooctylphenyl Ether (TritonX-100) (Nacalai Tesque, 2598785)
Blocking One-P (Nacalai Tescue, 05999-84)
DBCO-PEG5-NHS Ester (Click Chemistry Tools, A102P-2)
2-Amino-2-hydroxymethyl-1,3-propanediol (Tris) (Wako, 204-07885)
N-(2-Hydroxyethyl)piperazine-N'-2-ethanesulfonic Acid (HEPES) (Nacalai Tesque, 1751415)
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic Acid (TAPS) (Nacalai Tesque, 0894182)
0.5 M Ethylenediamine-N,N,N',N'-tetraacetic Acid (EDTA) solution (Dojindo, MB01)
Sodium Chloride (NaCl) (Ncalai Tesque, 3132005)
Sodium Hydrogen Carbonate (NaHCO3) (Nacali Tesque, 3121315)
Sodium Dodecyl Sulfate (SDS) (Wako, 191-07145)
Dithiothreitol (DTT) (Wako, 042-29222)
Dimethyl Sulfoxide (DMSO) (Wako, 041-29351)
N,N-dimethylformamide (DMF) (Nacalai Tesque, 1301694)
1 M Magnesium chloride (MgCl2) solution (Nacalai Tesque, 2094234)
Glycerol (Nacalai Tesuque, 1703865)
Spermidine (Wako, 195-09821)
Mouse anti-H3K4me3 (CMA304 16H10)²¹
Mouse anti-H3K27me3 (CMA323 1E7)²²
Mouse anti-H3K27ac (CMA309 9E2H10)²¹
Rabbit anti-CTCF (Abcam, ab70303)
Rabbit anti-MyoD (Santa Cruz ,sc-760, M-318)
AffiniPure Donkey Anti-Mouse IgG (H+L) (Jackson Immuno Research, 715-005-150)
AffiniPure Donkey Anti-Rabbit IgG (H+L) (Jackson Immuno Research, 711-005-152)
Tn5 (in house)²³
T4 DNA Ligase (NEB, M0202L)
T4 DNA Polymerase (NEB, M0203L)
T7 RNA Polymerase (Thermo Fisher Scientific, AM2085)
Ribonucleoside 5'-Triphosphates (NTP) (Takara, 4041, 4042, 4043, 4044)
Recombinant RNase Inhibitor (Takara, 2313A)
Recombinant DNase I (RNase-free) (Takara, 2270A)
SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara, 634891)
Agencourt AMPure XP beads (Beckman Coulter, A63881)
E-Gel Size Select II Agarose Gels, 2% (Thermo Fisher Scientific, G661012)
DNA Standards for Library Quantification (Takara, 638325)
Oligo DNAs (Table 1)
PD MiniTrap G-25 (GE Healthcare, 28918007)
Amicon Ultra-0.5 ml Centrifugal Filters 10k-off (Merck Millipore, UFC501096)
Amicon Ultra-0.5 ml Centrifugal Filters 100k-off (Merck Millipore, UFC510096)
Microcon Ultracel DNA Fast Flow Membrane (Merck Millipore, MRCF0R100)
Magnetic rack for AMPure XP beads
MinElute PCR Purification Kit (Qiagen, 28004)
RNeasy MinElute Cleanup Kit (Qiagen, 74204)
Agilent High Sensitivity DNA Kit (Agilent, 5067-4626)
Library Quantification Kit (Takara, 638324)

Equipment

Cell culture standard consumables
µ-Plate 96-well TC (ibiTreat) (ibidi, ib89626)
Protein LoBind 1.5 ml tube (Eppendorf, 0030108116)
0.2 ml 8-strip PCR tube (Thermo Fisher Scientific, N8010580, N8010535)
Fluorescence (confocal) Microscope
E-Gel Electrophoresis system (Thermo Fisher Scientific, G6512ST)
Nanodrop (Thermo Fisher Scientific)
qPCR (Agilent, MX300p)
Bioanalyzer 2100 (Agilent)
Centrifuge (Eppendorf)
Thermal cycler (Eppendorf)
Rotator (Taitec)
Heat block incubator (Taitec)

Reagents Setup

• 2x Tn5 dialysis buffer

100 mM HEPES-KOH (pH7.2)

200 mM NaCl

0.2 mM EDTA

20% Glycerol

0.2% TritonX-100

2 mM DTT 1 M 20 μl

  Aliquot 1-2 ml each, store at -30° C

• 5x TAPS-DMF buffer

50 mM TAPS-NaOH (pH8.5)

25 mM MgCl2

50% DMF

  Aliquot 1-2 ml each, store at -30° C

• 10x T7 RNA Polymerase buffer

400 mM Tris-HCl (pH8.0)

80 mM MgCl₂

20 mM Spermidine

50 mM DTT

  Aliquot 1-2 ml each, store at -30° C

• DBCO-PEG5-NHS Ester

Suspend 2 mg with 100 μl of DMSO (28.8 mM), aliquot 20 μl each and store at -30° C

• 10x annealing buffer

100 mM Tris-HCl (pH7.4)

10 mM EDTA

1 M NaCl

  Store at room temperature

• Antibody labeling with DBCO-PEG5-NHS Ester

Mix antibody with DBCO-PEG5-NHS Ester in a Protein LoBind tube

conjugation is performed with a molar ratio of about 1:10

IgG 100 μg

NaHCO₃ (1 M, pH8.3) 10 μl

DBCO-PEG5-NHS Ester (28.8 mM) 0.2 μl

PBS upto 100 μl

↓

Incubate the sample at room temperature for 1 h by rotating the tube

During this step, equilibrate the a PD MiniTrap G-25 desalting column with 2.5 ml of PBS, three times

↓

Apply the sample into the column

Add 500 μl of PBS and discard the flow through

↓

Place the column onto a new 1.5 ml tube

Add 500 μl of PBS and recover the flow through

↓

Transfer the sample into an Amicon Ultra-0.5 NMWL 10kDa centrifugal filter

Centrifuge at 14,000 xg for 20 min at 4° C

↓

Recover the sample (~25 μl) into a new 1.5 ml tube

Add ~50 μl of PBS

↓

Measure the concentration of IgG and DBCO using Nanodrop

molar extinction coefficiency of DBCO = 12,000 M⁻¹ cm⁻¹ at 309 nm
Typically approximately three DBCO per antibody

↓

Dilute to 1 mg/ml IgG with PBS

Store at 4 ° C

• ChILT DNA

Mix oligo DNAs in a 0.2 ml PCR tube

ChILT primer_Fw-azido (100 μM) 10 μl

ChILT primer_Rv-Ph,TMR (100 μM) 10 μl

10x annealing buffer 10 μl

Nuclease-free water 70 μl

Total 100 μl

↓

Thermal Cycler: incubate 95° C 5 min → lowering the temperature by 0.1oC/s to 20oC

Store at -30° C

• Oligonucleotide conjugation with antibody

Mix DBCO-PEG5 labeled-IgG with ChILT DNA in a Protein LoBind tube

conjugation is performed with a molar ratio of 1:2

DBCO-PEG5 labeled-IgG (1 mg/ml) 75 μl

ChILT DNA (annealed, 10 μM) 100 μl

↓

Incubate the sample at 4° C for 1 week by rotating the tube

Cu-free click reaction with azido-DNA is much slower than classical click reaction

↓

Apply the sample into a Microcon Ultracel DNA Fast Flow Membrane

Add 400 μl of PBS

Centrifuge at 500 xg for 20 min at 4° C

↓

After discarding the flow through, add 400 L of PBS

Centrifuge at 500 xg for 20 min at 4° C

Repeat this step 4 times

↓

Transfer the sample (~100 μl) into an Amicon Ultra-0.5 NMWL 100kDa centrifugal filter Centrifuge at 14,000 xg for 20 min at 4° C

↓

Recover the sample (~25 μl) into a new 1.5 ml tube

Adjust the volume to 85 μl (two-fold concentrated) with PBS

Store at 4° C

ChILT work flow

(Day 1) Cell sorting, plating

Plate cells on a 96-well ibidi plate

Single cell by cell sorter

≥100 cells by manual

↓

Incubate at 37° C in a humidified atmosphere of 5% CO₂ overnight

↓

(Day 2) immunostaining

After removing the medium, add 100 μl of 1% formaldehyde/DMEM

Incubate at room temperature for 5 min, without shaking

↓

After removing the formaldehyde, wash the cells with 200 μl of PBS

↓

After removing PBS, add 150 μl of 1% Triton/PBS

Incubate at room temperature for 20 min with gentle shaking

↓

After removing Triton, wash the cells with 200 μl of PBS

↓

After removing PBS, add 100 μl of Blocking One-P

Incubate at room temperature for 20 min with gentle shaking

↓

After removing Blocking One-P, wash the cells with 200 μl of PBS

↓

After removing PBS, add 100 μl of primary antibody (2 μg/ml) in 0.1x Blocking One-P/PBS

Incubate at room temperature for 6 h with gentle shaking

↓

After removing the primary antibody, wash the cells with 200 μl of PBS

Gently shaking at room temperature for 5 min, three times

↓

Plate on ice

↓

After removing PBS, add 100 μl of ChILT probe (1 μg/ml) in 0.1x Blocking One-P, 0.5 M NaCl/PBS (ice-cold)

Incubate at 4° C overnight with gentle shaking

↓

(Day 3) ChILT reaction

After removing the ChILT probe, wash the cells with 200 μl of PBS (ice-cold)

Gently shaking at 4° C for 20 min, three times

↓

After removing PBS, add 50 μl of Tn5 (90 ng/well; 0.883 mg/ml 0.1 μl/well) in 1x Tn5 dialysis buffer

Incubate at room temperature for 10 min with gentle shaking

↓

Without removing Tn5, add 50 μl of MEDS-B (10 μM, 0.1 μl/well) in 1x Tn5 dialysis buffer

Incubate at room temperature for 1 h with gentle shaking

↓

After removing the supernatant, wash the cells with 100 μl of PBS

Gently shaking at room temperature for 5 min, three times

↓

After removing PBS, wash the cells with 100 μl of 1x Tn5 dialysis buffer

↓

After removing the buffer, add 100 μl of 1x TAPS-DMF buffer

Incubate at 37° C for 1 h with gentle shaking

↓

After removing the buffer, add 100 μl of 0.2% SDS

Incubate at room temperature for 10 min, without shaking

↓

After removing SDS, wash the cells with 100 μl of PBS, three times, without interval

↓

After removing PBS, wash the cells with 100 μl of 1x T4 DNA Ligase buffer

↓

Prepare fill-in solution (according to the number)

Nuclease-free water 88.5 μl

10x T4 DNA Ligase buffer 10 μl

dNTP mix (10 mM) 0.5 μl

T4 DNA ligase 0.5 μl

T4 DNA Polymerase 0.5 μl

Total 100 μl

After removing the buffer, add 100 μl of fill-in solution

Incubate at room temperature for 30 min with gentle shaking

↓

After removing the supernatant, add 100 μl of 0.2% SDS

Incubate at room temperature for 10 min, without shaking

↓

After removing SDS, wash the cells with 100 μl of PBS, three times, without interval

↓

After removing PBS, wash the cells with 100 μl of 1x T7 RNA Polymerase buffer

↓

Prepare in situ transcription solution (according to the number)

Nuclease-free water 80 μl

10x T7 RNA Polymerase buffer 10 μl

25 mM each NTP 8 μl

RNase Inhibitor 1 μl

T7 RNA Polymerase 1 μl

Total 100 μl

After removing the buffer, add 100 μl of in situ transcription solution

Seal the wells with parafilm

Incubate at 37° C overnight with gentle shaking

↓

(Day 4) library preparation: SMART-seq v4

Add 1 μl of DNaseI

Incubate at 37° C for 30 min with gentle shaking

↓

Purify the ChILT RNA by RNeasy MinElute Cleanup Kit

Recover the supernatant into a 1.5 ml tube

↓

Add 350 μl of Buffer RLT into the well and transfer this rinsed fraction to the same tube

↓

Add 250 μl of EtOH and mix well by pipetting up and down

↓

Transfer the sample into RNeasy MinElute Spin Column

Centrifuge at 13,000 rpm for 1 min

↓

Place the column in a new collection tube

Add 500 μl of Buffer RPE

Centrifuge at 13,000 rpm for 1 min

↓

After discarding the flow through, add 500 μl of 80% EtOH

Centrifuge at 13,000 rpm for 2 min

↓

After discarding the flow through, centrifuge again at 13,000 rpm for 1 min with the lid opened

↓

Place the column on a new 1.5 ml tube and add 10 μl of Nuclease-free water directly to the membrane

After waiting for 1 min, centrifuge at 13,000 rpm for 1 min

↓

Recover the flow through and add again to the membrane

After waiting for 1 min, centrifuge again at 13,000 rpm for 1 min

↓

SMAT-seq v4 using custom primers

• Primer annealing

Prepare the reaction buffer (according to the number, for 10 samples below)

10x Lysis buffer 4.75 μl

Read2 primer (12 μM) 5 μl

RNase Inhibitor 0.25 μl

Total 10 μl

Mix ChILT RNA and the reaction buffer in a 0.2 ml 8-strip PCR tube

RNA Sample 5.25 μl

Reaction buffer 1 μl

Total 6.25 μl

↓

Thermal Cycler: incubate 72° C 3 min → on ice

↓

• First-strand cDNA Synthesis

Prepare the master mix (according to the number)

5x Ultra Low First-Strand Buffer 2 μl

SMART-Seq v4 Oligonucleotide (48 μM) 0.5 μl

RNase Inhibitor 0.25 μl

SMART Scribe Reverse Transcriptase 1 μl

Total 3.75 μl

Add the master mix into the RNA sample

RNA sample 6.25 μl

Master mix 3.75 μl

Total 10 μl

↓

Thermal Cycler: incubate 42° C 1.5 h → 70° C 10 min → 4° C

• cDNA Amplification

Prepare the master mix (according to the number)

2x SeqAmp PCR Buffer 12.5 μl

Ad1 primer (12 μM) 0.5 μl

SeqAmp DNA Polymerase 0.5 μl

Nuclease-free water 1.0 μl

Total 14.5 μl

Mix ChILT cDNA, Ad2 primer and the master mix in a new 0.2 ml 8-strip PCR tube

First-strand cDNA 10 μl

Ad2 primer (12 μM) 0.5 μl

Master mix 14.5 μl

Total 25 μl

↓

Thermal Cycler: 95° C 1 min → (98° C 10 sec → 65° C 30 sec → 68° C 3 min) →72° C 10 min → 4° C

↓

(Day 5) library purification

Incubate the AMPure XP beads at room temperature and mix well by vortexing before use

Add 10x Lysis buffer and AMPure XP beads to the amplified ChILT DNA

Amplified DNA Sample 25 μl

10x Lysis buffer 0.5 μl

AMPure XP beads 25 μl

Total 50 μl

Mix well by pipetting up and down and incubate for 5 min

↓

Place on a magnetic rack and wait for 5 min

↓

Discard the supernatant (45 μl)

↓

Wash the beads with 200 μl of 80% EtOH, twice

After incubating for 30 sec, discard the supernatant

↓

Wait for ≦5 min. Take care not to over dry

↓

Outside the rack,

Add 27 μl of EB buffer (Qiagen) and mix well by pipetting up and down

Incubate for 2 min

↓

Place on the rack and wait for 5 min

↓

Recover the supernatant (25 μl) into a new 0.2-ml PCR tube

↓

Repeat again the purification with 25 μl of AMPure XP beads

10x Lysis buffer is not required in second purification

↓

Recover the elute (25 μl) into a new tube

↓

option Pool and purify the samples by MinElute PCR Purification Kit

Mix the samples and adjust the volume to 100 μl with Nuclease-free water

↓

Add 500 μl of Buffer PB and mix well by pipetting up and down

↓

Transfer the sample into a MinElute column

Centrifuge at 13,000 rpm for 1 min

↓

After discarding the flow through, add 700 μl of Buffer PE

Centrifuge at 13,000 rpm for 1 min

↓

After discarding the flow through, centrifuge again at 13,000 rpm for 1 min with the lid opened

↓

Place the column on a new 1.5 ml tube and add 25 μl of Buffer EB

After waiting for 1 min, centrifuge at 13,000 rpm for 1 min

↓

• Size selection using E-Gel Electrophoresis system with E-Gel Size Select II agarose gel

Recover 250-350 bp fragments

↓

Purify the size selected samples by MinElute PCR Purification Kit

Add 5 volumes of Buffer PB to 1 volume of the sample and mix well

↓

Transfer the sample into MinElute column

Centrifuge at 13,000 rpm for 1 min

↓

After discarding the flow through, add 700 μl of Buffer PE

Centrifuge at 13,000 rpm for 1 min

↓

After discarding the flow through, centrifuge again at 13,000 rpm for 1 min with the lid opened

↓

Place the column on a new 1.5 ml tube and add 10 μl of Buffer EB

After waiting for 1 min, centrifuge at 13,000 rpm for 1 min

↓

• Quantification of the ChILT library

Bioanalyzer (DNA high sensitivity kit)

qPCR (TaKaRa Library Quantification Kit)

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T.H., A.H., Y.O. and H.K. are involved in a pending patent related to ChIL technology.

supplement0.xlsx
Table.1 ChIL oligo DNAs Table.1 ChIL oligo DNAs

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Detailed protocol ─ Chromatin Integration labeling

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Detailed protocol ─ Chromatin Integration labeling

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Abstract

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Introduction

Reagents

Equipment

Procedure

References

Additional Declarations

Supplementary Files

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Terms of Service

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