Illumina HiSeq 4000 paired end sequencing; HNF4A is required to specify glucocorticoid action in the liver - ChIPseq
HNF4A is required to specify glucocorticoid action in the liver - ChIPseq
Mice were treated acutely with an intraperitoneal injection of dexamethasone or vehicle, and liver tissue harvested one hour later.  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