Illumina HiSeq 4000 paired end sequencing; Mouse white adipose tissue HaloREVERBα ChIP-seq
Mouse white adipose tissue HaloREVERBα ChIP-seq
For experimental samples, gonadal white adipose tissue was collected from male homozygous HaloReverbα mice at ZT8; for control samples, tissue was collected from male homozygous HaloReverbα mice at ZT20, and from male WT mice (related colony) at ZT8. REVERBα HaloChIP-seq was performed using a protocol based on (Hunter et al., 2020 – doi 10.1073/pnas.2005330117), with modifications informed by (Castellano-Castillo et al., 2018 – doi 10.1371/journal.pone.0192314). Reagents were from the ChIP-IT High Sensitivity Chromatin Preparation kit (Active Motif 53046) and the HaloCHIP System (Promega  G9410), unless otherwise specified. Freshly-harvested gonadal white adipose tissue was finely minced and dual-crosslinked in 10ml 0.5M disuccinimidyl glutarate (ThermoFisher 20593) followed by 10ml 1% formaldehyde-PBS. After quenching with glycine, tissue pieces were washed x2 in ice-cold ChIP wash buffer (Active Motif), then resuspended in 5ml freshly-made adipose lysis buffer (10mM Tris HCl, 140mM NaCl, 5mM EDTA, 1% NP-40) supplemented with a HaloChIP-compatible protease inhibitor cocktail (Promega G6521). Fixation and wash solutions were changed by centrifuging tissue suspensions at 1250g for 3 minutes (4ºC), and removing the infranatant from below the floating adipose tissue pieces with glass Pasteur pipettes. Tissue was disrupted mechanically with the Qiagen TissueRuptor, then incubated on ice for 30 minutes to lyse cells. Following centrifugation (2780g for 3 minutes, 4ºC), the lipid layer and supernatant were carefully removed, and the nuclei pellet resuspended in 650ul Mammalian Lysis Buffer (Promega), supplemented with protease inhibitor cocktail. Suspensions were once again incubated on ice (15 minutes) prior to low-amplitude probe sonication (Active Motif) (x8 2 minute cycles of 30s on/30s off, 20% amplitude). Cellular debris was pelleted by spinning at 21,000g for 3 minutes (4ºC), and 600ul of the chromatin suspension added to HaloLink™ Resin, prepared as per manufacturer's instructions. Pull-down reactions were rotated for 3 hours at room temperature, subsequent to wash and elution steps as per manufacturer's HaloCHIP System instructions. Eluted ChIP DNA was purified with the ChIP DNA Clean & Concentrator kit (Zymo D5205). ChIP DNA was quantified with the Qubit dsDNA HS Assay Kit (Invitrogen Q32851). For each ChIP-seq library preparation, ChIP DNA from 3-7 mice was pooled to total 5ng ChIP DNA. ChIP DNA pools (x2 ZT8 HaloReverbα pools, x1 ZT20 HaloReverbα pool, x1 ZT8 WT pool) were submitted to the University of Manchester Genomic Technologies Core Facility for TruSeq ChIP library preparation (Illumina) as per manufacturer's instructions.