GSM2486811: HUVEC TCC hypoxia replicate 1; Homo sapiens; Hi-C
90 million human umbilical vein endothelial were fixed with 1 % PFA/PBS in room temperature for 10 minutes and crosslinking was stopped by incubating cells 5 min in presence of 125 mM glycine. Cells were washed two times with ice-cold PBS containing 0.5 % BSA and scraped into lysis buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 5 mM MgCl2, 0.2 % NP-40 and protease inhibitor cocktail (Roche). Samples were vortexed, incubated on ice for 10 min, centrifugated for 5 minutes in 1500 g in +4 °C and washed once again with lysis buffer. Cells were washed twice with ice-cold wash buffer containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl and 0.5M EDTA and nuclei were isolated by incubating samples 10 minutes at +65 °C in wash buffer with 0.55 % SDS and collected by sentrifugation (1000 g for 1 min). Later steps followed the protocol described (Kalhor et al., 2012), except MboI (NEB) was used to digest chromatin, T1 Streptavidin dynabeads (Invitrogen) were used in all the pulldown steps, NucleoSpin Gel & PCR Clean-up kit (Macherey-Nagel) for clean-up steps and Bioruptor NextGen sonicator for DNA shearing (High setting, 30s/30s cycles for total 35 cycles). Libraries were amplified in 15 PCR cycles, size selected for 225 – 550 bp range on 10 % TBE-gel (Invitrogen). Gel was grinded and soaked with 0.1 % Tween-20 and 150 mM NaCl and DNA was eluted through Ultrafree Centrifugal filter column (Millipore), cleaned up with ChIP DNA clean & concentrator kit (Zymo Research) and paired-end sequenced with Illumina HiSeq 2000.