GSM4455323: H3K27Ac.Y.NR.8.2; Mus musculus; ChIP-Seq
Liver was collected at the indicated time points and processed for ChIP-seq as previously published. Briefly, liver was fixed in disuccinimidyl glutarate (2mM in PBS + 1% DMSO, 30 min) and formaldehyde (1% in PBS, 10 min) prior to aliquoting and freezing at -80˚C. For BMAL1 (antibody produced in Bass Lab as in (43), Millipore ABE2599) and HSF1 (Cell Signaling 4356) ChIP-Seq, nuclei from a quarter of a liver were isolated in buffer (150 mM NaCl, 5 mM EDTA pH8, 50 mM Tris-HCl pH8, 0.35% NP-40) in the presence of protease inhibitors by needle, then sheared in 1% SDS buffer in a sonicator (diagenode) for 6 cycles 30on/30off at 4˚C. An aliquot for input samples was taken after shearing. Chromatin was diluted 1:10 in dilution buffer (.01% SDS, 1.1% Triton X-100, 167 mM NaCl, 1.2 mM EDTA pH8, 1.67 mM Tris-HCl pH8), and immunoprecipitations with 15 µg of antibody were performed overnight at 4˚C, rotating. Secondary-conjugated, BSA-blocked paramagnetic beads were used to pull down protein/DNA complexes. Chromatin was de-crosslinked and purified by Qiagen MinElute column. Inputs were quantified by Qubit Fluorometer (Invitrogen) for each liver, and 2 ng of equimolar pools of the replicates per condition were processed into libraries. Library prep with NEBNext Ultra II Library prep kit. Libraries were size-selected to 200-600 bp on the Sage Pippin Prep prior to PCR amplification. Average library size and concentration were determined by Bioanalyzer and qPCR (NEBNext Library Quant kit), respectively, prior to pooling.
GEO Accession
GSM4455323