ChIP-seq of Irx3 in primary cells from mouse gWAT and iWAT on days -1 and 1 of adipogenic differentiation

A map of global Irx3 binding sites during early stages of adipogenic differentiation. C57BL/gNJ (B6N) mice were fed a SDS maintenance chow diet, and at the age of 6-10 weeks, gWAT and iWAT was isolated and primary preadipocytes were isolated. For each depot, cells from 6-12 mice were pooled. Isolated cells were cultured until confluence in 2D and induced to differentiate 2 days post confluence by a standard adipogenic cocktail. One day before (day -1) and one day after (day 1) induction of differentiation, cells were fixated by formaldehyde, treated with protease inhibitors, snap-frozen and shipped to Active Motif for ChIP-seq analysis. Reads were aligned to mouse genome (mm10) using Bowtie2 (version 2.3.4.3), with duplicates and low quality mapping reads (<30) removed via samtools. Peak files were annotated using the R package ChIPseeker (version 1.22.0). Log2 ChIP-over-input tracks for each alignment were generated using deepTools bamCompare (version 3.1.2), and blacklisted regions from Encode were excluded. Bam files were submitted to Macs2 (version 2.1.1) for peak calling. A q-value of 1E-4 and 10X enrichment was used as threshold.