GSM4975490: 6h hypoxia input control; Mus musculus; ChIP-Seq
Chip was performed on snapfrozen liver samples derived from mice that were injected with PBS or 10 mg/kg DEX. 200mg of tissue was homogenized in PBS and then crosslinked with 2% formaldehyde in PBS for 20 min at RT while rotating. This reaction was stopped by adding 125mM glycine for 10min at RT. Tissue was then collected in ice-cold PBS and subsequently lysed in lysis buffer (0.1% SDS, 1% Triton X-100, 0.15M NaCl, 1mM EDTA and 20mM Tris pH8, supplemented with protease inhibitors). Lysates were sonicated at 4°C to yield 200-800bp DNA fragments. IP was performed on 100µl lysates diluted 1:3 in incubation buffer (0.15% SDS, 1% Triton X-100, 0.15M NaCl, 1mM EDTA and 20mM HEPES) with 5µg of rabbit anti-GR antibody (H300, sc-8992; Santa Cruz) or normal rabbit IgG (10500C; Invitrogen) as negative control at 4°C overnight. After two hours BSA blocked nProtein sepharose beads (GE Healthcare) were added to the lysates. The following day, beads were washed in WBI, WBII, LiCl buffer and twice in EDTA/HEPES buffer (0.5M EDTA and 1M HEPES). Chromatin was eluted in 200µl elution buffer (0,1M NaHCO3 and 10% SDS) supplemented with proteinase K. Next, proteins were decrosslinked by raising the incubation temperature to 65°C for 16h and DNA purified using the PCR purification kit (Qiagen), and eluting in 50µl of Qiagen Elution Buffer Fifteen ng of purified DNA was used to generate ChIP-seq libraries according to manufacturer's protocol using NEBNext Ultra II DNA Library Prep Kit (E7645L, NEB).