GSM8594817: ZT18 Dex Rep2; Mus musculus; ChIP-Seq
Tissue was collected after 1 hour. Chromatin was prepared from flash-frozen liver tissue using the Active Motif ChIP-IT High Sensitivity kit (Active Motif). 25μg of chromatin was incubated overnight at 4C with a GR antibody cocktail (ProteinTech 24050-1-A (lot 00044414) and Cell Signaling D8H2 (lot 2)). To permit direct comparison between samples, and to control for technical variation between ChIP reactions, a spike-in ChIP normalisation strategy was employed, with 30ng spike-in chromatin (53083, Active Motif) and 2μg spike-in antibody (61686, Active Motif (lot 34216004)) being included in each IP reaction. To obtain sufficient DNA for next-generation sequencing, three IP reactions were carried out for each sample, and then pooled for the pull-down step. Antibody was pulled down using washed magnetic protein G agarose beads (ReSyn Biosciences). Beads were washed five times with AM1 Wash Buffer (Active Motif) then DNA eluted as per ChIP-IT kit instructions. ChIP-seq DNA was purified with the MinElute PCR Purification kit (Qiagen). Illumina TruSeq ChIP Library Preparation Kit, according to the manufacturer's protocol Sequenced paired end 76bp on HISEQ4000 at the Genomic Technologies Core Facility University of Manchester