GSM5930912: 22Rv1_HiC_rep2; Homo sapiens; Hi-C
In situ Hi-C experiments were performed in 22Rv1 cells, using a previously described protocol 25 with minor modifications. Briefly, 1× 107 cells were digested and cross-linked in 1% formaldehyde. Chromatin was digested with 150 U MboⅠ (NEB, R0147) at 37 ℃ with rotation. To fill in restriction fragment overhangs, 50 U DNA Polymerase Ⅰ, Large Fragment (NEB, M0210), combined with dNTPs containing biotin-dATP (Thermofisher, 19524016), were added and incubated at 37℃ for 1 h. For ligation, 4000 U T4 DNA ligase (NEB, M0202) was added and incubated at room temperature for 4 h with slow rotation. To reverse cross-links, 25 μl proteinase K (20 mg/ml) was added and incubated at 65 ℃ overnight. Purified Hi-C samples were sheared to 200-300 bp by sonication. Biotin pull-down was performed using Streptavidin C-1 beads (Life Technologies, 65002) with 2× biotin binding buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA and 2 M NaCl). The Hi-C library was amplified with 8 cycles using Illumina primers.