GSM6234768: Hi-C, MCF7, 1; Homo sapiens; Hi-C
Three million cells were fixed with freshly made 1% formaldehyde for 10 min at room temperature for each sample. The reaction was quenched by adding 2 M glycine to a final concentration of 0.2 M for 5 min at room temperature. Fixed cells were lysed in lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl and 0.2% IGEPAL CA-630 with proteinase inhibitor) for 1h at 4 °C with rotation. The supernatant was discarded and the pellet was gently resuspended in 1 × CutSmart Buffer (NEB, B7204) containing 0.1% SDS and incubated at 65°C for 10 min. Permeabilization was quenched by adding 10% Triton X-100 and incubated at 37°C for 1h. Chromatin was digested overnight at 37 °C with rotation by adding the DpnII restriction enzyme (NEB, R0543M). Digested chromatin ends were filled and marked with biotin-14-dATP (Thermo Fisher, 19524016) using a large Klenow fragment of DNA polymerase I (NEB, M0210) for 1h at 37 °C. Samples were then diluted and ligated using T4 DNA ligase (Thermo Fisher, EL0013) at 16 °C overnight. Chromatin was de-cross-linked, and proteins were removed by proteinase K, followed by phenol/chloroform extraction coupled with isopropanol precipitation using glycogen as a carrier. Biotin-14-dATP from unligated DNA ends was removed by T4 DNA polymerase (NEB, M0203) at 12 °C for 2 h. DNA was purified by phenol/chloroform and sonicated using a Covaris S220 (Covaris, USA) to approximately 300-600 bp. Biotinylated DNA fragments were then pulled down using Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher, 65001). The DNA ends were subsequently repaired and phosphorylated using T4 DNA polymerase (NEB, M0203), Klenow fragment of DNA polymerase I (NEB, M0210), and T4 Polynucleotide Kinase NK (NEB, M0201). The DNA was further prepared for sequencing using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme Biotech, ND607) and barcoded adaptors were ligated using TruePrep Index Kit V2 for Illumina (Vazyme Biotech, TD202). Each Hi-C library was indexed uniquely and amplified by 12 cycles of PCR for all samples. PCR purification and size selection was carried out using the VAHTS DNA Clean Beads (Vazyme Biotech, N411). Library quantity was assessed using a Qubit 3.0 Fluorometer (Invitrogen, USA). For each individual, the library was sequenced with 150 bp paired-end reads on the Illumina HiSeq X Ten platform.