GSM1315483: BV2-ChIPseq-notx-CEBPb-pCDH; Mus musculus; ChIP-Seq
20x10^6 cells were crosslinked in Formaldehyde/PBS 1% for 10 min at RT. After quenching the reaction by adding 125mM glycine, cells were washed 2X with PBS and were centrifuged (8 min, 800g, 4°C). Cells were resuspended in swelling buffer (10mM HEPES/KOH pH 7.9, 85mM KCl, 1mM EDTA, 0.5% IGEPAL CA-630, 1X protease inhibitor cocktail (Roche), 1mM PMSF) for 5 min. Cells were spun down and resuspended in 500μl lysis buffer (50mM Tris- HCl pH 7.4, 1% SDS, 0.5% EmpigenBB, 10mM EDTA, 1X protease inhibitor cocktail (Roche), 1mM PMSF) and chromatin was sheared to an average DNA size of 300–400bp by administering 5 pulses of 10 sec duration at 10 W power output with 30 sec pause on ice using a Misonix 3000 sonicator. The lysate was cleared by centrifugation (5 min, 16000g, 4°C), and supernatant was diluted 2.5-fold with 750μl dilution buffer (20mM Tris-HCl pH 7.4, 100mM NaCl, 0.5% Triton X-100, 2mM EDTA, 1X protease inhibitor cocktail (Roche)). The diluted lysate was pre-cleared by rotating for 2h at 4°C with 120μl 50% rProtein A sepharose Fast Flow (GE Healthcare). The beads were discarded, and 1% of the supernatant were kept as ChIP input. The protein of interest was immunoprecipitated by rotating the supernatant with 2.5 μg antibody overnight at 4°C, then adding 50μl blocked rProtein A sepharose and rotating the sample for an additional 1h at 4°C. The beads were pelleted (2 min, 1000g, 4°C), the supernatant discarded, and the beads were transferred in 400μl wash buffer I (WBI) (20mM Tris-HCl pH 7.4, 150mM NaCl, 0.1% SDS, 1% Triton X-100, 2mM EDTA) into 0.45 μm filter cartridges (Ultrafree MC, Millipore), spun dry (1 minute, 2200g, 4°C), washed one more time with WBI, and twice each with WBII (20mM Tris-HCl pH 7.4, 500mM NaCl, 1% Triton X-100, 2mM EDTA), WBIII (10mM Tris-HCl pH 7.4, 250mM LiCl, 1% IGEPAL CA-630, 1% Na-deoxycholate, 1mM EDTA), and TE. Immunoprecipitated chromatin was eluted twice with 100 μl elution buffer each (100mM NaHCO3, 1% SDS) into fresh tubes for 20 min. Eluates were pooled, the Na+ concentration was adjusted to 300mM with 5M NaCl and crosslinks were reversed overnight at 65°C in a hybridization oven. The samples were sequentially incubated at 37°C for 2h each with 0.33mg/ml RNase A and 0.5mg/ml proteinase K. The DNA was isolated using the QiaQuick PCR purification kit (Qiagen). DNA from chromatin immunoprecipitation (10–50ng) was adapter-ligated and PCR amplified according to the manufacturer's protocol (Illumina).