GSM7897979: Wild Type Replicate 1 HiC; Homo sapiens; Hi-C
Hi-C libraries were prepared using in situ Hi-C protocol as previously described (Rao et al., 2014) with some modifications. In brief, 0.2 to 0.5 million HeLa cells were crosslinked with 16% formaldehyde (Thermo 28908) to a final concentration of 1% for 10 min at room temperature, followed by quenched by 0.2 M glycine for 5 min. Cell nuclei were extracted with lysis buffer (10 mM Tris-HCl pH8.0, 10 mM NaCl, 0.2% Igepal CA630) mixed with 50× protease inhibitor (Sigma P8340) on ice for at least 15 min. Cell pellets were resuspended in 0.5% SDS (prepared from 10% SDS  (Sigma 71736)) and incubated at 62°C for exactly 10 min, then quenched with 1.5% trix-100 (freshly prepared from 10% Triton X-100 (Sigma 648463)) for 15 min at 37°C. DNA was then digested with Dpn II restriction enzyme (NEB R0543L) for 16h at 37°C, followed by an incubation at 62°C for 20 minutes. End repair of DNA as well as biotin labeling was achieved by incubation at 37° for 1h in the fill-in master mix (with a final concentration of 0.4mM biotin-14-dATP (Invitrogen 19524016), 3.3 mM dCTP, 3.3 mM dGTP, 3.3 mM dTTP, 5000U/mL DNA Polymerase I, Large (Klenow) Fragment (NEB M0210L)). DNA was then ligated by incubation in a ligation master mix (composed of 664ul of water, 120μl of 10X T4 DNA ligase buffer (NEB B0202), 100μl of 10% Triton X-100 (Sigma 71736), 6μl of 20mg/ml Bovine Serum Albumin (Sigma B8667), and 10μl of 400 U/ μl T4 DNA Ligase (NEB M0202)) at room temperature with low-speed rotation for 4h. Cell pellets were resuspended in 300 μL of PK digestion buffer (10 mM Tris-HCl pH8.0, 0.5 M NaCl, 1% SDS), followed by the addition of 50 μL of 20mg/ml proteinase K (QIAGEN 19133). The mixture was digested at 55°C for 30 minutes to uncross-link the DNA, and then incubated overnight at 68°C. To efficiently purify the DNA, the TIANamp Genomic DNA Kit (TIANGEN DP304) was used instead of low-temperature precipitation with ethanol and sodium acetate. Sonication was then employed to break DNA into an average size of 300-500 bp. Biotin-labeled DNA was pulled down with Dynabeads MyOne Streptavidin C1 beads (Invitrogen 65001) and prepped for library construction. Libraries were constructed using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme ND607) following the instructions.