GSM8741975: HiC data for COLO320-DM cells; Homo sapiens; Hi-C
Cells were first cross-linked with 1% formaldehyde at room temperature for 10 minutes, followed by quenching with 0.125M glycine for 5 minutes. The cells were then subjected to a secondary cross-linking with 3 mM EGS for 40 minutes, and the cross-linking was quenched with 0.4M glycine for 10 minutes. The doubly cross-linked cells were washed with PBS containing 0.05% bovine serum albumin and permeabilized with 0.05% digitonin at room temperature for 15 minutes. They were subsequently treated with 0.1% SDS at 65 °C for 10 minutes, and SDS was neutralized at 37 °C with 1.8% Triton X-100 for 15 minutes. Chromatin DNA was then digested at 37 °C for 2 hours using 50 U each of MboI and DdeI (NEB), followed by DNA end filling with 15 U Klenow (NEB) and dNTPs at 37 °C for 1 hour. DNA ligation was performed at 16 °C for 4 hours using 50 U T4 DNA Ligase (NEB) to ligate adjacent DNA regions. After washing the cells twice in wash buffer, they were incubated overnight with diluted primary antibody in a Dig-wash buffer containing 2 mM EDTA. The next day, the secondary antibody was added and incubated at room temperature for 1 hour. The cells were washed twice in Dig-Wash buffer to remove unbound antibodies. The pG-Tn5 adapter complex was prepared in Dig-300 buffer and incubated with the cells at room temperature for 1 hour, followed by two washes to remove unbound pG-Tn5. The cells were then resuspended in Tagmentation buffer and incubated at 37 °C for 1 hour. Tagmentation was terminated by incubation at 55 °C for 1 hour in a solution containing 16 mM EDTA, 0.1% SDS, and 0.16 mg/mL protease K. DNA was extracted using phenol-chloroform-isoamyl alcohol and ethanol precipitation. Library amplification was performed by PCR: 3 minutes at 72 °C, 30 seconds at 98 °C, followed by 16 cycles of 15 seconds at 98 °C, 30 seconds at 60 °C, and 30 seconds at 72 °C, with a final 3-minute extension at 72 °C. The amplified DNA was purified using Ampure XP beads, and the libraries were sequenced on the Illumina NovaSeq platform.