GSM8641650: HCT116 p53-/- WT [HiC]; Homo sapiens; Hi-C
HiC-seq for cell lines was performed based on HiC2.0 protocol (Belaghzal et al., 2017) with slight modifications. Briefly, cells were crosslinked with 1% formaldehyde for 10 minutes and quenched with 140 mM glycine. Five million crosslinked cells were lysed with 10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 0.2% NP-40. Pelleted nuclei were lysed in 0.1% SDS and quenched by 1% Triton X-100, followed by digestion with DpnII (NEB, no. R0543) at 37 C overnight in ThermoMixer with constant mixing at 600 RPM. Digested DNA ends were then labeled with biotin-14-dATP (Invitrogen, no. 19524016) by DNA polymerase I, large (Klenow) fragment (NEB, no. M0210) at 37 C, 1 hour. Labeled DNA was ligated by T4 DNA ligase (Invitrogen, no. 15224017) and incubated with Proteinase K (Invitrogen, no. 25530015) to reverse crosslinking. Resulting DNA was purified and sonicated by Covaris to produce DNA fragments ~100-400bp. The biotin-labeled DNA fragments were then enriched by MyOne Streptavidin C1 beads (Invitrogen, no. 65001). DNA fragments while on beads were end repaired and made with NEBNext UltraII DNA library prep kit (NEB, no. E7645) with NEBNext Adaptor and Index (PCR amplified 9 cycles). Lastly the libraries were size selected by AMpure XP beads and sequenced on Illumina NovaSeq S4 lane at Novogene. Quality check at various steps was performed according to the HiC2.0 protocol.