title: Primary mouse hepatocytes, GR ChIP-seq, 1h GW7647 treatment (0.5 microM)
design: Glucocorticoid Receptor ChIP-seq in primary mouse hepatocytes upon 1h stimulation. Samples from 6 independent immunoprecipitations, obtained using 2 biological replicates were pooled. Each biological replicated was obtained by pooling hepatocytes from 3 mice.     Cell pellets were lysed with 500 μl of lysis buffer (0.1% SDS, 1% Triton X-100, 0.15M NaCl, 1mM EDTA, 20mM Tris pH 8) supplemented with protease inhibitor cocktail (Pierce, cat. 88265) and sonicated 30x (30s on/ 30s off, high intensity) using a Bioruptor sonicator (Diagenode) at 4°C. Insoluble material was removed by centrifugation (30 min, 20000g). For each immunoprecipitation 100 μl of chromatin was diluted 4 times with incubation buffer (0.15% SDS, 1% Triton X-100, 0.15M NaCl, 1mM EDTA, 20mM HEPES), mixed with antibodies (anti-GR (Santa Cruz, H-300), anti-PPARα (Santa-Cruz, H-98) and incubated for 2h at 4°C with rotation. Protein A Sepharose beads (GE Healthcare Life Sciences, cat. 17-5138-01) were washed 2x and blocked in incubation buffer with 1 μg/μl of BSA (2h, 4°C with rotation). Diluted chromatin samples were mixed with a blocked Protein A Sepharose solution and incubated overnight at 4°C with rotation. Next day, beads were washed twice in wash buffer 1 (0.1% SDS, 0.1% NaDOC, 1% Triton X-100, 0.15M NaCl, 1mM EDTA, 20mM HEPES), once in wash buffer 2 (0.1% SDS, 0.1% NaDOC, 1% Triton X-100, 0.5M NaCl, 1mM EDTA, 20mM HEPES) and buffer 3 (0.25M LiCl, 0.5% NaDOC, 0.5% NP-40, 1mM EDTA, 20mM HEPES) and twice in wash buffer 4 (1mM EDTA, 20mM HEPES). Protein-DNA complexes were eluted in 200μl (1% SDS + 0.1M NaHCO3) at room temperature and cross-links were reversed at 65°C overnight by adding 8μl of 5M NaCl, together with 8μl of RNAse A (0.5 mg/ml, Macherey-Nagel, cat. 740505) and 10 μl of Proteinase K (20 mg/ml, Qiagen, cat. 19131). DNA was purified with a QIAQuick PCR purification kit (Qiagen, cat. 28106) according to the user’s manual.