GSM5906057: iWAT_BMAL1-1; Mus musculus; ChIP-Seq
NuTRAP nuclei and ribosomal RNA were isolated according to established methods (PMID 28122230). Brown adipose and inguinal white adipose depots were snap frozen in liquid nitrogen, minced, dounce homogenized, spun down and FACS sorted to isolate for ATAC-Seq. Tissues were minced, homogenized, ribosomal RNA was affinity purified using anti-GFP antibodies and protein G Dynabeads and extracted using the Qiagen Micro RNeasy kit. For ChIP-seq experiments, minced inguinal WAT was fixed in disuccinimidyl glutarate (2mM in PBS + 1% DMSO, 30 min) and  formaldehyde (1% in PBS, 10 min). Nuclei 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 dounce homogenization, then  sheared in 1% SDS buffer by sonication using a Covaris E220 (5% duty factor, 140 W power, 200 cycles  per burst, 4 minute total shearing time, 4°C). An aliquot for input samples was taken after shearing.  Chromatin was diluted 1:10 in dilution buffer (0.01% SDS, 1.1% Triton X-100, 167 mM NaCl, 1.2 mM  EDTA pH 8, 1.67 mM Tris-HCl pH 7.4). Immunoprecipitation was performed using 15 μg of BMAL1  antibody (Millipore ABE2599) at 4°C with rotation. Protein A paramagnetic beads (ThermoFisher) were  used to pull down protein/DNA complexes. Chromatin was de-crosslinked and purified by QIAGEN  MinElute column prior to library prep with NEBNext Ultra II DNA library prep kit. Inputs were  quantified by Qubit Fluorometer (Invitrogen), pooled, and 2 ng were processed into libraries. Libraries  were size-selected to 200-600 bp on the Sage Pippin Prep, amplified and barcoded using 13 cycles of  PCR, and sequenced using single-end 75 bp sequencing on a NextSeq 500 to an average depth of ~30M  reads. ATAC-seq libraries were constructed according to the Omni-ATAC protocol (PMID 28846090). RNA-Seq libraries were constructed from 250 ng of RNA using the NEBNext RNA Ultra Directional library prep kit. ChIP-seq samples were de-crosslinked and purified by QIAGEN  MinElute column prior to library prep with NEBNext Ultra II DNA library prep kit. Inputs were  quantified by Qubit Fluorometer (Invitrogen), pooled, and 2 ng were processed into libraries. Libraries  were size-selected to 200-600 bp on the Sage Pippin Prep, amplified and barcoded using 13 cycles of  PCR.