GSM8161060: chip, adf_f_2; Mus musculus; ChIP-Seq
Liver pieces (150 mg) were cross-linked with phosphate-buffered saline (PBS) containing 2mM disuccinimidyl glutarate (DSG, Santa Cruz Biotechnology, cat# sc-285455). Livers were homogenized with a Dounce homogenizer and rotated for 30 min at room temperature. Crosslinked samples were washed in phosphate buffered saline (PBS), resuspended in ChIP lysis (0.5% SDS, 10mM EDTA, 50mM Tris-HCl pH8) buffer and sonicated to release 100-1000 bp fragments. Antibody against PPARa was conjugated to magnetic beads for 2 h at 4°C. Chromatin was immunoprecipitated with antibody-bead conjugates for 16 h at 4°C. Immunocomplexes were washed sequentially with the following buffers:low salt buffer (0.01% SDS, 1% Triton x-100, 2 mM EDTA, 20 mM Tris-HCl pH 8, 150 mM NaCl), high salt buffer (0.01% SDS, 1% Triton x-100, 2 mM EDTA, 20 mM Tris-HCl pH 8, 500 mM NaCl), LiCl buffer (0.25M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid, 1mM EDTA, 10 mM Tris pH 8.1) and twice with TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8). Chromatin was eluted, de-crosslinked for 4 h at 65°C and deproteinized with proteinase K (Hy Labs, cat# EPR9016) for 1 h at 50°C..DNA was subsequently isolated using MinElute DNA purification kit. ChIP DNA libraries were prepared using the KAPA HyperPrep Kit (KAPA biosystems, cat# KR0961).