GSM3931972: BMAL1-LSKO.NMN.6.2; 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, nucleii 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.