GSM4008547: Liver, INPUT_ZT14 (DNA input); Mus musculus; ChIP-Seq
The nuclei isolation was performed as described in (Ripperger and Schibler, 2006), except that the ultracentrifugation was performed for 1h at 24,000 rpm (100,000g) at 1°C in a Beckmann SW28 rotor. The nuclei were lysed with 20 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA and 1% SDS. The sonication of the chromatin was performed with a Branson SLPe sonicator during 15 cycles of 10 sec at 50% amplitude. The sheared chromatin was then diluted 10x in 50 mM Hepes pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.5 mM PMSF, and protease inhibitor cocktail (Sigma, #P8340).The inputs were isolated and the immunoprecipitation performed with a homemade anti-RXR antibody, generously provided by C Rochette-Egly, overnight at 4°C on a rotating platform. Protein A-sepharose CL-4B beads (GE Healthcare, #17-0780-01) were added and the tubes were kept rotating for 2 h at 4°C. The beads were then washed 2x with 50 mM Hepes pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, 0.5 mM PMSF, and protease inhibitor cocktail (Sigma, #P8340); 2x with 50 mM Hepes pH 7.9, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, 0.5 mM PMSF, and protease inhibitor cocktail (Sigma, #P8340); 2x with 20 mM Tris pH 8, 1 mM EDTA, 250mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 0.5 mM PMSF, and protease inhibitor cocktail (Sigma, #P8340); and 2x with 10 mM Tris pH 8 and 1 mM EDTA. The elution of the chromatin from the beads was done twice with 50 mM Tris pH 8, 1 mM EDTA, 1% SDS and 50 mM NaHCO3 for 10 min at 65°C. NaCl was added to 200 mM and the chromatin was decrosslinked at 65°C overnight. The samples were treated with 25 µg/ml of RNase A for 1 h at 37°C, then with 5 mM of EDTA and 50 µg/ml of proteinase K for 2 h at 42°C. The DNA was purified with the NucleoSpin® Gel and PCR Clean-up from Macherey-Nagel. Nuclei isolation method as described in Nature Genetics by (Ripperger and Schibler, 2006). The DNA concentration was measured with the Qubit® Fluorometer (Invitrogen). The libraries were prepared starting from 2.1 ng to 4.2 ng of IP samples and 8 ng of inputs samples, using the Microplex Library Preparation Kit (Diagenode, #C05010010)