title: GSM6016405: HICHIP-22RV1-C1-CTCF; Homo sapiens; OTHER
library_construction_protocol: HiChIP was performed as previously described with a few modifications. Ten million cells were collected and pelleted and resuspended in 1% formaldehyde for 10 min with rotation at room temperature. The crosslinking was then quenched in 125 mM glycine for 5 min. The cells were washed twice by PBS and then resuspended in 500 μL ice-cold Hi-C lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2% NP-40, 1× protease inhibitor) followed by 30 min rotation at 4 °C. Nuclei were pelleted by 2500 rcf centrifugation for 5 min at 4 °C followed by washing once in Hi-C lysis buffer. The pelleted nuclei were resuspended in 100 μL 5% SDS and incubated at 62 °C for 10 minutes. To quench the SDS, 335 μL 1.5% Triton X-100 was then added and the mixture was incubated at 37 °C for 15 minutes. The chromatin was digested by adding 50 μL NEB buffer 2 and 375 U MboI restriction enzyme (NEB, R0147) and incubating at 37 °C for 20 minutes. MboI restriction enzyme was then inactivated by 62 °C incubation for 10 minutes. The fill-in master mix (1.5 μL of 10 mM dTTP, 1.5 μL of 10 mM dCTP, 1.5 μL of 10 mM dGTP, 37.5 μL of 0.4 mM biotin-dATP, 10 μL of 5U/μL DNA Polymerase I (NEB, M0210) was added and the tube was incubated at 37 °C for 1 hours with rotation. The ligation mix, containing 10 μL 400 U/μL T4 DNA Ligase (NEB, M0202), 150 μL 10X NEB T4 DNA ligase buffer with 10 mM ATP (NEB, B0202), 125 μL 10% Triton X-100, 3 μL 50 mg/mL BSA, 660 μL H2O, was added and the tube was incubated at room temperature for 4 hours with rotation. The nuclei was pelleted and then resuspended in 880 μL nuclear lysis buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS, 1× protease inhibitor). Nuclei was sonicated in a water bath sonicator (Diagenode bioruptor) to generate chromatin fragments at 300~800 bp. Immunoprecipitation was performed by adding protein A beads and appropriate antibodies  to sheared chromatin before incubation at 4 °C overnight with rotation. The ChIPed DNA was eluted from beads by 200 μL ChIP elution buffer (50 mM NaHCO3, 1% SDS) and purified by Zymo DNA Clean & Concentrator kit. The biotin-labelled DNA was captured by streptavidin C1 beads (Invitrogen, 65001) and subjected to tagmentation using 2.5 μL Tn5 (Illumina) for 50 ng DNA. The HiChIP DNA was amplified by ~8 cycles using primers containing Illumina sequencing linkers. Each HiChIP library was sequenced by an Illumina NovaSeq system to generate ~150 million 2 ×150 bp paired-end reads.