GSM5145773: H2_HiC; Homo sapiens; Hi-C
5e6 cells were crosslinked with 1% formaldehyde for 10 minutes at room temperature. The reaction was quenched for 5 minutes at room temperature with a final concentration of 200 mM glycine. Fixed cells were lysed in 1mL cell lysis buffer (10mM Tris-HCl pH 8, 10mM NaCl, 0.2% NP-40/Igepal) for 30 minutes on ice. The cell suspension was pelleted at 2500 x g for 5 minutes at 4oC and washed once with lysis buffer. The cell pellet was resuspended in 0.5% SDS and incubated at 62oC for 10 minutes. 10% Triton X-100 was used to quench the SDS and incubated at 37oC for 15 minutes. 100U of MboI was used to digest the chromatin overnigt at 37oC with shaking. Following heat inactivation of MboI, restriction fragment overhangs where marked with biotin. Ligation of marked ends was performed using T4 ligase at room temperature for 4 hours. Ligated DNA was sheared using a Covaris M220 with the power settings of peak incident power of 50, duty cycle 200, 200 cycles/burst, for 90 seconds. Sheared DNA was purified using AMPure XP beads (Beckman Coulter, A63881). Biotin-labelled DNA was isolated using Dynabeads MyOne Streptavidin C1 beads Libraries were prepared using standard reagents from New England BioLabs for Illumina sequencing