title: GSM3456997: BRCA1_WT2 Hi-C; Homo sapiens; Hi-C
library_construction_protocol: Harvested cells were washed once with PBS, fixed with 2% formaldehyde for 10 min in PBS at room temperature, quenched with 0.2M glycine for 5 min. After washing once with PBS, pelleted cells were flash frozen and stored at -80°C for further processing. Nuclei were isolated with lysis buffer (10mM Tris-HCl (pH 8.0), 10mM NaCl, 0.2% IGEPAL CA630) with incubation on ice for 5 min, washed once same lysis buffer. Pelleted nuclei were permeabilized with 0.5% SDS for 10 min at 62°C, quenched with Triton X-100 for 15 min at 37 °C. Chromatin was digested with a 4 cutter restriction enzyme (MboI, 100U, NEB) overnight at 37°C with mixing. After inactivating digestion at 62°C for 20 min, the 5' overhangs were filled in with biotinylated-14-dATP (Life Tech) and Klenow (40U, NEB) for 90 min at 37°C with mixing. Biotinylated ends were ligated with T4 DNA ligase (2000U, NEB) for 4 hours at room temperature with mixing. DNA was reverse crosslinked with Proteinase K (400mg, NEB) and 1% SDS for 30 min at 55 °C, followed by an overnight incubation at 68°C with NaCl. DNA was purified with ethanol precipitation, and sheared on an ultrasonicator (Covaris S220; duty cycle: 10; intensity: 4; cycles/burst: 200; duration: 55s; number of cycles: 1) to a fragment range of 300-700 bp. Double size selection was performed using SPRI beads (Beckman Coulter). Biotinylated fragments were enriched by pulldown with Dynabeads MyOne T1 Streptavidin beads (Life Technologies), followed by library preparation (Quick Ligation Kit, NEB). Final library was amplified by PCR (Phusion DNA Polymerase, Thermo). Libraries were sequenced on HiSeq4000 Paired-End for 100bp (Illumina).