GSM6206661: HiC_ESC_WT_Rep1; Mus musculus; Hi-C
To extract the nuclei, cells were incubated on ice for 10 min with ice-cold Lysis Buffer (50mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM 788 EDTA, 0.5% NP-40, 1.15% Triton X-100, 25X Protease Inhibitor in Milli-Q Water). Extracted nuclei were centrifuged at 750g for 5 min at 4°C, two times washed with 1X DPBS, snap frozen and stored at -80°C. Nuclei were first permeabilized using 0.5% SDS at 62°C for 10 min and later, chromatin was digested with DpnII (NEB, #R0543) for 90 min at 37°C with gentle rotation. The overhangs generated by digestion were filled and marked with biotin-14-dATP (Thermo, #19524016) by a 90 min incubation at 37°C with gentle rotation. After, DNA fragments were ligated for 4 hours at 20°C with gentle rotation using T4 DNA Ligase (Thermo, #M0202M). Chromatin was then reverse-crosslinked, precipitated and sheared with Covaris S220 (2 cycles, each 50sec long; 10% duty; 4 intensity; 200 cycles/burst). The biotin-marked-DNA shared fragments were pulled down using Dynabeads MyOne Streptavidin T1 beads (Thermo, #65601), purified and further processed for Illumina sequencing with NEBNext Ultra II Library Prep Kit for Illumina according to the kit guidelines (NEBNext End Prep, Adaptor Ligation, PCR enrichment of Adaptor-Ligated DNA using NEBNext Multiplex Oligos for Illumina). Clean up and size selection were performed with AMPure beads.