GSM4891198: CLP_PBS-2; Mus musculus; ChIP-Seq
50mg 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)   fter 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 TruSeq ChIP Library Preparation Kit