GSM6326543: A001C007 [Hi-C]; Homo sapiens; Hi-C
5-10 mg of snap-frozen tissue was loaded in tissueTUBE-TT05 (Covaris 520071) and cryopulverized using the Covaris CP02 cryoPREP Automated Dry Pulverizer following manufacturer's procedure. Pulverized tissues were treated with freeze substitution40 by submerging in 1ml -80 oC 0.01% formaldehyde (ThermoFisher 28906), 97% ethanol, and 2% water. Samples were incubated in dry ice for 3 hours on a rotor with spinning speed around 100 rpm, and then placed in a CoolCell Container (Corning), transferred to a -20 oC freezer for overnight incubation. On day 2, the Container was transferred to a 4 oC cold room, spinning on a rotor around 100 rpm for 1 hour to bring the sample temperature above freezing point. The tissue samples were separated from ethanol solution by centrifuging at 300 g for 5 min in a 4 oC microcentrifuge. Crosslinking was performed by incubating with 1ml 1% TBS-formaldehyde for 10 min at room temperature. The solution was quenched by adding 80 μl 2.5 M glycine and incubated for 5 min. Samples were centrifuged, washed once with 1 ml TBS (pH 7.5), and resuspended with 250 μl Hi-C lysis buffer (10 mM Tris-HCl, pH8.0, 10 mM NaCl, 0.2% Igepal CA630) plus 50 μl proteinase inhibitor cocktail (Sigma P8340). Nuclei extraction was performed on ice by squeezing the samples with 1.5 ml disposable pellet pestles (Fisher Scientific 12-141-368) for 15-20 times. The crude suspension was centrifuged at 1500 g, 4 oC for 5 min, resuspended in 800 μl Hi-C lysis buffer and passed through a 100 μm strainer (Sysmex). After centrifugation, purified nuclei were resuspended in 170 μl 10 mM Tris-HCl containing 0.5% Triton X-100 (Sigma 93443), and incubated at room temperature with rotation for 15 min. Ten microliter of 1% SDS, 20 μl Cutsmart buffer (NEB), 3 μl HinP1I (NEB R0124S), 3 μl DdeI (NEB R0175L), 3 μl CviAII (NEB R0640L), 3 μl FspBI (ThermoFisher ER1762), and 0.6 μl MseI (NEB R0525M), were added to the suspension in the indicated order. The mixture was incubated at 25 oC and then 37 oC for 2 hours each with rotation. To stop restriction digestion, the suspension was incubated in a 62 oC heating block for 20 min followed by cool down. End repair was carried out by adding a 30 μl solution containing 0.5 mM biotin-14-dATP (Active Motif 14138), 0.5 mM biotin-14-dCTP (AAT bio 17019), 0.5 mM dTTP, 0.5 mM dGTP, and 4 μl Klenow DNA polymerase (NEB M0210L) to the mixture, and incubated for 1 hour at 37 oC with rotation. For ligation, a 750 μl solution containing 1x NEB T4 DNA ligase buffer (NEB B0202), 120 μg BSA (ThermoFisher AM2616), and 2000 U T4 DNA ligase (NEB M0202M) was added. The mixture was incubated at room temperature for 90 min, then 4 oC overnight, and then room temperature for 60 min with rotation.To perform reverse crosslinking, the mixture was centrifuged at 1500 g for 5 min. Supernatant was replaced with a mixture of 300 μl 1x T4 ligase buffer, 30 μl 20 mg/ml proteinase K (ThermoFisher 25530049), 30 μl 10% SDS, and 40 μl 5 M NaCl. The suspension was then incubated at 66 oC for 4 hours. DNA content was then purified in 20 μl 10 mM Tris-HCl by phenol-chloroform extraction.To generate mHi-C sequencing library, 300 ng of purified DNA was tagmented with 2.5 μl Tn5 transposase (APExBIO K1155, discontinued) loaded with equimolar Mosaic Ends containing Illumina Nextera i5 and i7 extensions following the manufacturer's protocol. Tagmentation was performed in a 100 μl buffer containing 10% DMF, 10 mM Tris-HCl, and 150 mM NaCl, at 55 oC for 10 min. The product was column purified (Zymo D4014) and PCR amplified for 2 cycles using the NEBNext master mix (NEB M0544L) with Illumina Nextera primers and conditions. Biotin enrichment was then performed by adding 20 μl Dynabeads MyOne Streptavidin C1 (ThermoFisher 65001) and incubating at room temperature for 30 min with rotation. The magnetic beads were washed three times with 1x wash buffer (10 mM Tris-HCl pH7.5, 1 mM NaCl, 0.5 mM EDTA) and one time with 10 mM Tris-HCl. Final libraries were obtained by amplifying the beads with additional 8 cycles of PCR, followed by purification with SPRI (Beckman B23318) size selection at 0.5x-1.1x range. The 33 samples were combined to 2 pools and sequenced with 2 NovaSeq (Illumina) S4 200 cycle flow cells. multi-RE digested in situ Hi-C