GSM8617899: T47D, Hypoxia, WT, Flag, replicate 4; Homo sapiens; ChIP-Seq
1×107 of T47D cells were fixed using 1% formaldehyde for 10 minutes and quenched with 125 mM glycine for 5 minutes (cells treated under hypoxia were fixed in hypoxia chamber). Post-fixation, cells were washed twice with cold PBS supplemented with protease inhibitors. Cells were lysed using LB1 buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) followed by LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and LB3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine) buffers. Nuclei were collected and sonicated using Bioruptor sonicator (15 cycles, 30 s on and 30 s off). After Triton X-100 treatment (1% final concentration), supernatants were collected post-centrifugation (20,000g, 10 min, 4 °C) and incubated with antibody-bounddynabeads for approximately 8 hours at 4 °C. Following washing, chromatin-protein complexes bound to beads were eluted, crosslinks reversed overnight at 65 °C, and treated with RNase (1 hour at 37 °C) and protease K (2 hours at 55 °C). DNA was recovered using PCR purification kit and subjected to library preparation using the NEBNext Ultra II kit, following the manufacturer's instructions.