snHiChew

Zhichao Chen

Jan 12, 2025

snHiChew

This protocol is a draft, published without a DOI.

Zhichao Chen¹

¹BGI

Zhichao Chen

BGI

Protocol Citation: Zhichao Chen 2025. snHiChew. protocols.io https://protocols.io/view/snhichew-dxma7k2e

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: January 12, 2025

Last Modified: January 12, 2025

Protocol Integer ID: 118146

Abstract

We present HiChew (High efficient chromatin conformation capture with post-enrichment), a novel method for analyzing three-dimensional chromatin organization in single cells and low-input samples. HiChew combines efficient sticky-end ligation with
post-PCR enrichment using methylation-based selection, addressing key limitations of existing chromatin conformation capture methods.

Cell Type and Cell Culture

NIH/3T3, HEK293T and CTCF Knockdown 293T were cultured in DMEM (Gibco, Cat. No. 11965092) medium supplemented with 10%FBS (Gibco, Cat. No. 10099141) and 1%penicillin–streptomycin (Gibco Cat. No. 15140122), under the conditions of 37°C, 5%CO_2. For transcript inhibition experiment, HEK293T cells were treated with 1 μM triptolide for 2 hours. 

Purification of spermatogenic cells was performed as previously description{MicroRNAs control mRNA fate by compartmentalization based on 3′ UTR length in male germ cells | Genome Biology | Full Text}. Briefly, after collection and decapsulation, two testes were digested in EKRB buffer containing collagenase to disperse the testicular cells. The dispersed cells were washed, digested with trypsin and DNase I, and washed three times. Cells were resuspended in  1×PBS solution and processed to fixation steps.

Cell Fixation

Culture medium of HEK293T cells was removed, and cells were washed once with 1×PBS. Trypsinization was carried out by adding 2 mL of 0.025% trypsin to a 10-cm culture dish, and incubated at 37°C until detachment. Detached cells were collected in 4 mL of complete DMEM medium and centrifuged at 400 × g for 3 minutes.     

The cell pellet was washed twice with 1×PBS, and fixed with 2 mL of 1% formaldehyde (16% wt/vol, ThermoFisher, Cat. No. 28908) in PBS at room temperature for 10 minutes. Fixation was terminated with 100 μL of 2.5 M glycine for 5 minutes. 

Cells were centrifuged at 800 × g for 5 minutes, washed twice with 1×PBS, and resuspended in 2 mL of PBS containing 3 mM ethylene glycol bis(succinimidyl succinate) (EGS, Thermo, Cat. No. 21565) for a second fixation at room temperature for 45 minutes. Fixation was terminated by adding 400 μL of 2.5 M glycine to quench the reaction, and the tube was incubated for 5 min at room temperature, followed by centrifugation and washing twice with 1×PBS. 

Fixed cells were either processed immediately or flash-frozen in liquid nitrogen for storage.

Cell Lysis

Cells were lysed in 500 μL of  permeabilization buffer (10 mM Tris-HCl [pH 8.0], 10 mM NaCl, 0.2% CA-630, 1× protease inhibitors cocktail) per 2 × 10^6 cells and incubated on ice for 10 minutes. 

After centrifugation at 800 × g at 4°C for 3 minutes, the supernatant was discarded, and the pellet was washed once with 1.2×NEBuffer r3.1. 

The pellet was resuspended in 199 μL of 1.2× NEBuffer r3.1, supplemented with 1 μL of 20% SDS (final concentration 0.1%), and incubated at 65°C for 10 minutes. 

After cooling on ice for 2 minutes, 10 μL of 20% Triton X-100 was added to neutralize SDS, followed by incubation at 37°C for 15 minutes.

Enzyme Digestion and Ligation

For restriction digestion, 200 U of DpnII (NEB, Cat. No. R0543L) was added to the reaction and incubated at 37°C overnight. Aliquots were de-crosslinked with proteinase K at 65°C for 1h and analyzed by 1% agarose gel electrophoresis to confirm digestion efficiency. 

Digested samples were washed twice with washing buffer (1×PBS, 1 mM EDTA, 1 mM EGTA, 0.1% Triton X-100) and resuspended in 195 μL of 1×T4 DNA ligase buffer containing 0.1% BSA. Ligation was carried out by adding 1 μL of T4 DNA ligase (2000U/μL) (ABclonal, Cat. No. RK21500) to a final concentration of 10 U/μL and incubating at 16°C overnight. Aliquots were de-crosslinked with proteinase K at 65°C for 1h and analyzed by 1% agarose gel electrophoresis to confirm ligation efficiency.

Enzyme Digestion and dA-tailing

Cell nuclei were washed twice with 1.1× rCutSmart buffer (New England Biolabs, Cat. No. B7204S) and resuspended in 200 μL of the same buffer. For digestion, 10 μL of HpyCH4V enzyme (NEB, Cat. No. R0620L) was added, and the reaction was incubated at 37°C with shaking at 1,000 rpm for 4 hours. Aliquots were de-crosslinked with proteinase K at 65°C for 1h and analyzed by 1% agarose gel electrophoresis to confirm digestion efficiency.

Following digestion, cell nuclei were pelleted by centrifugation at 500 × g for 3 minutes, the supernatant was removed, and nuclei were washed twice with washing buffer (1× PBS, 1 mM EDTA, 1 mM EGTA, 0.1% Triton X-100). 

Nuclei were then resuspended and counted using a cell counting plate. Approximately 5 × 10⁵ nuclei were transferred to a new 1.5 mL centrifuge tube, and 25 μL of dA-tailing reaction buffer and 10 μL of Klenow fragment (New England Biolabs, NEBNext dA-Tailing Module, Cat. No. E6053L) were added. The reaction volume was adjusted to 250 μL with nuclease-free water, and the mixture was incubated at 37°C with shaking at 1,000 rpm for 90 minutes.

After dA-tailing, 200 μL of reaction termination buffer (1× PBS, 50 mM EDTA, 50 mM EGTA, 0.1% Triton X-100) was added, and the nuclei were centrifuged at 800 × g for 2 minutes. Nuclei were washed twice with washing buffer, resuspended in 200 μL of the same buffer, and filtered through 20-μm filter.

The nuclei concentration was determined using a cell counting plate, and 2 × 10⁵ nuclei were resuspended in 1,165 μL of BSA buffer (1× PBS, 0.1% Triton X-100, 0.3% BSA).

Index Adapter Ligation

Custom-designed 96 index primers, avoiding the GATC sequence (5’- CGA CCA CCG AGA TGT ACA C - 6 bp index - TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG T - 3’ and 5’ - /phos/CTG TCT CTT ATA CAC ATC TCC GAG CCC ACG AGA C - 3’) were used to prepare 50 μM index-Y adapters. 

Each well of a 96-well plate was loaded with 2 μL of index adapter. 

The nuclei solution from step 17 was well-mixed before loading 11.6 μl of nuclei solution into each well of a 96-well plate in step 19.

For ligation, 220 μL of 2 × Instant Sticky-End Ligase Master Mix (New England Biolabs, Cat. No. M0370), 352 μL of 5 × Quick Ligase Buffer (New England Biolabs, Cat. No. B6058S), and 132 μL of 1,2-propanediol (Sigma-Aldrich, Cat. No. 398039) were mixed and 6.4 μL of mixture was distributed into each well of the plate containing the nuclei and adapters. The reaction was incubated at 20°C with shaking at 1,600 r.p.m. for 3 hours, with intermittent shaking for 30 seconds every 5 minutes.

After ligation, 50 μL of reaction termination buffer was added to each well, mixed, and incubated at room temperature for 15 minutes. The products from all wells were pooled into a 15 mL centrifuge tube. 

The combined nuclei were centrifuged at 800 × g for 5 minutes, washed twice with washing buffer, and resuspended in BSA buffer. Single cells were filtered through a 10-μm filter and counted using a cell counting plate. 

A total of 930-1,400 nuclei were resuspended in 350 μL of BSA buffer, and 3 μL of the suspension was distributed into each well of a new 96-well plate, with approximately 800 - 1,200 nuclei in total across all wells.

Reverse Crosslinking and PCR Amplification

For reverse crosslinking, Qiagen protease (Qiagen, Cat. No. 19157) was prepared at 0.1 U/mL in EB buffer, and 3 μL was added to each well. Plates were mixed by shaking, centrifuged briefly, and incubated in a thermocycler at 50°C for 1 hour, 65°C for 2 hours, and 70°C for 30 minutes. 

Next, 0.6 μL of a 10 μM i7 PCR primer (5′-CAA GCA GAA GAC GGC ATA CGA GAT - 8bp index - GTC TCG TGG GCT CGG-3′) was added to each well. The PCR reaction was prepared with 760 μL of 2× KAPA HiFi ready mix (KAPA Biosystems, Cat. No. KK2602), 65 μL of HiChew-P5 primer (5′-/phos/AAT GAT ACG GCG ACC ACC GAG ATG TAC AC-3′), and 135 μL of nuclease-free water. 

A total of 9.3 μL of the reaction mix was added to each well of the plate contaning the decrosslinked DNA, and amplification was performed for 12 -15 cycles on a PCR thermocycle instrument. 

After amplification, the PCR products from all wells were pooled into a 15 mL centrifuge tube and purified using the QIAGEN PCR purification kit (Qiagen, Cat. No. 28104). Part of the PCR product was processed to library circulation  and sequencing to produce Dip-C data. 

Methylation Marking

Purified DNA (1 μg) was methylated at GATC sites by incubating with 5 μL of 10× dam Methyltransferase Reaction Buffer, 0.25 μL of 32 mM S-adenosylmethionine (SAM), and 1 μL of dam Methyltransferase (NEB, Cat. No. M0222L) in a total reaction volume of 50 μL. The reaction was carried out at 37°C for 1 hour, followed by purification with 1×AMPure XP beads (Beckman Coulter, Cat. No. A63882).

Antibody Preparation for IP

Ten microlitre of Protein A/G Magnetic Beads (Thermo, Cat. No. 88802) per sample were washed twice with PBST (1×PBS, 0.1% Tween 20) and blocked with 50 μL of SuperBlock Blocking Buffer (Thermo, Cat. No. 37580) for 15 minutes at room temperature.

Beads were washed twice with 1×IP buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% CA-630, 1 mM EDTA) and resuspended in 48 μL of 1×IP buffer.

 To coat the beads, 2 μL of Anti-N6-methyladenosine (m6A) antibody (Merck, Cat. No. ABE572-I-100UG) was added, and the mixture was incubated overnight at 4°C with rotation.

Immunoprecipitation

For IP, 500ng of methylated DNA containing 20 μL of 10 μM blicking primer mix (P5 primer (5′-AAT GAT ACG GCG ACC ACC GAG ATG TAC AC-3′), P7 primer (5′-CAA GCA GAA GAC GGC ATA CGA G-3′), R1N primer (5′-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG-3′), R2N primer(5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA G-3′)) was filled to 40 μL with water and denatured at 95°C for 5 minutes, chilled on ice for 2 minutes, and then mixed with 10 μL of 5×IP buffer (50 mM Tris-HCl [pH 7.5], 750 mM NaCl, 2.5% CA-630, 5 mM EDTA) and 50 μL of antibody-coated beads. The mixture was incubated at 4°C with rotation for 2 hours. 

Beads were washed sequentially: once with medium-stringency RIPA buffer (10 mM Tris-HCl [pH 8.0], 300 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.2% SDS, 0.1% sodium deoxycholate), twice with high-stringency RIPA buffer (10 mM Tris-HCl [pH 8.0], 350 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.23% SDS, 0.1% sodium deoxycholate), and twice with 1×IP buffer. 

DNA was eluted by incubating beads with 22 μL of EB buffer containing 0.05 U/mL Qiagen protease at 50°C for 30 minutes. The supernatant was collected and incubated at 70°C for 15 minutes to inactivate the protease.

Post-IP PCR Amplification

For PCR amplification, 30 μL of a reaction mixture containing 25 μL of 2×KAPA HiFi readymix (KAPA Biosystems), 1.5 μL of HiChew-P5 primer (5′-/phos/AAT GAT ACG GCG ACC ACC GAG ATG TAC AC-3′) and 1.5 μL of P7 primer (5′-CAA GCA GAA GAC GGC ATA CGA G-3′), and 2 μL of nuclease-free water was added to the eluted DNA. Amplification was performed for 6–7 cycles using a thermocycler, followed by purification with 1×AMPure XP beads.

Library Circulation and Sequencing

Library circularization was performed using the MGIEasy Circulating Kit (MGI, Cat. No. 1000005259) according to the manufacturer’s protocol. 

The circularized library was processed with the MGISEQ-2000RS High Throughput Sequencing Reagent Kit (PE100) (MGI, Cat. No. 1000012554) to generate DNA nanoballs (DNBs). 

Sequencing was carried out on the MGISEQ-2000 platform to generate paired-end 100 bp reads.

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