title: GSM7777927: floxed mice fasted for 24h followed by refeeding of 24h_2; Mus musculus; ChIP-Seq
library_construction_protocol: Liver pieces (150 mg) were cross-linked with phosphate-buffered saline (PBS) containing 2mM disuccinimidyl glutarate (DSG, Santa Cruz biotechnologies cat# sc-285455). Livers were homogenized with a Dounce homogenizer and rotated for 30 min at room temperature. Samples were spun down and pellet was crosslinked again with PBS containing 1% formaldehyde (Electron Microscopy Sciences, cat# 15714). After 10 min, samples were quenched with 0.125 M glycine. Samples were then washed with PBS, resuspended in ChIP lysis buffer (0.5% SDS, 10mM EDTA, 50mM Tris-HCl pH8) and sonicated (Bioruptor Plus, Diagenode) to release 100–1000 bp fragments. Samples were diluted 1:5 with ChIP dilution buffer (170 mM NaCl, 17 mM Tris-HCl pH8, 1.2 mM EDTA, 1.1% Triton x-100, 0.01% SDS). Antibody against LXRα (R&D Systems cat# PP-PPZ0412-00, 4 µg per sample), was conjugated to magnetic beads (Sera-Mag, Merck, cat# GE17152104010150) 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 pH8, 150 mM NaCl), high salt buffer (0.01% SDS, 1%Triton x-100, 2 mM EDTA, 20 mM Tris-HCl pH8, 500 mM NaCl), LiCl buffer (0.25M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid, 1mM EDTA, 10mM Tris pH8.1) and twice with TE buffer (10 mM Tris-HCl, 1 mM EDTA pH8). 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 (Qiagen cat# 20-28006). ChIP DNA libraries were prepared using the KAPA HyperPrep Kit (KAPA biosystems, cat# KR0961).