GSM8462686: Chipseq_Lig4mm_Rad21_Auxin_rep2; Homo sapiens; ChIP-Seq
Chipseq: Cultured cells were fixed with 1% formaldehyde for 10 min at room temperature and the reaction was quenched with 125 mM glycine. Twenty million fixed cells were washed with PBS, snap-frozen and stored at -80°C until further processing. Before use, the cells were resuspended in 1 ml of RIPA buffer (10 mM Tris pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100 and freshly added Complete Mini EDTA free proteinase inhibitor. Sonication was performed using Branson sonicator at amplitude 35%, 12 cycles of 20 sec sonication followed by 30 sec of pause or using Covaris ME220 sonicator at duty factor 20%, peak incident power 75, cycle/burst 1000 for 6 min at 4°C. The antibody-protein solution was then incubated with 40 ml of Dynabeads Protein A for 40 min at room temperature under agitation. Antibody-bound beads were washed twice with RIPA buffer, twice with RIPA buffer containing 0.3M NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate), once with LiCl buffer (0.25M LiCl, 0.5% NP-40, 0.5% NaDOC), once with TE pH 8.0 containing 0.2% Triton X-100, and once with TE pH 8.0. Crosslinks were reversed by incubating the beads at 65°C for 4 hours in the presence of 0.3% SDS and 1 mg/ml Proteinase K. ChIP DNA was purified by ChIP DNA clean and concentrator column. Chipseq: Ovation Ultralow Library System V2 kit , Hi-C library were amplified directly from of the beads with 4-6 cycles of PCR, using Illumina primers (Bioo Scientific) and Q5 Hi-Fidelity DNA polymerase