Illumina HiSeq 4000 paired end sequencing; Tissue-selective targeting redefines the mechanism of nuclear receptor Reverbα control over clock and metabolism - ChIP-seq
Tissue-selective targeting redefines the mechanism of nuclear receptor Reverbα control over clock and metabolism - ChIP-seq
Liver tissue was collected from wildtype (n=2) and homozygous (n=3) HaloReverbα mice at ZT8 and from homozygous (n=2) HaloReverbα mice at ZT20, then flash-frozen in liquid nitrogen. A dual-crosslinking strategy was employed to capture tethered protein-protein-DNA interactions in addition to protein-DNA interactions. Frozen tissue was first homogenised in 2mM disuccinimidyl glutarate (DSG) (Thermo Fisher Scientific) using a handheld TissueRuptor (Qiagen) and rotated at room temperature for 15 minutes. After being spun down, the cell pellet was resuspended in 1% formaldehyde-PBS, rotated for a further 15 minutes, before glycine quenching with Stop Solution (Active Motif ChIP-IT High Sensitivity kit 53040). The cell pellet was washed and dounce-homogenised as per the standard Active Motif ChIP-IT kit instructions. It was then resuspended in 650μl of Mammalian Cell Lysis Buffer (Promega HaloChIP kit) supplemented with Protease Inhibitor Cocktail (Promega) and incubated on ice for 15 minutes before sonication at 20% amplitude for 8 cycles (30s on, 30s off, with 2 minutes of 'on' time per cycle), using the Active Motif EpiShear Probe Sonicator. Chromatin concentration was quantified with the Qubit BR dsDNA assay (Q32850, Thermo Fisher Scientific) and shearing efficacy checked with TapeStation electrophoresis (Agilent Technologies). For the pull-down step, 100μg chromatin, in a volume of 650μl Mammalian Cell Lysis Buffer, was rotated at room temperature with prepared HaloLink Resin (Promega) as per the Promega HaloChIP kit protocol. Wash and elution steps were also carried out as per the same manufacturer's protocol, incorporating the recommended LiCl buffer wash step. ChIP DNA was cleaned up with the MinElute PCR Purification Kit (Qiagen) and quantified with the Qubit HS dsDNA assay (Thermo Fisher Scientific). Samples were not pooled; each biological replicate was taken forward individually to library preparation. Illumina TruSeq ChIP Library Preparation Kit, according to the manufacturer's protocol
Experimental Factor: genotype
HaloReverb wildtype
Experimental Factor: time
8
Experimental Factor: immunoprecipitate
Halo ChIP-kit