Genome-wide GR occupancy and chromatin landscape in skeletal muscles

Purpose: The aim of this study is to identify the GR genome binding profile as well as that of Pol II, H3K27ac, H3K4me1, H3K4me3 and Ctcf in skeletal muscles. Methods: Libraries were prepared from immunoprecipitated DNA according to standard methods. ChIP-seq libraries were sequenced using a HiSeq 4000 (Illumina) and mapped to the mm10 reference genome using bowtie 2 (Langmead et al., 2009). Data were further analysed using the peak finding algorithm MACS 2 (Zhang et al., 2008) using input as control. All peaks with FDR greater than 0.1 % were excluded from further analysis. The uniquely mapped reads were used to generate the genome-wide intensity profiles, which were visualized using the IGV genome browser (Thorvaldsdottir et al., 2012). Results: HOMER (Heinz et al., 2010) was used to annotate peaks, to calculate overlaps between different peak files, and for motif searches. The genomic features (promoter, exon, intron, 3' UTR, and intergenic regions) were defined and calculated using Refseq and HOMER. Genes annotated by HOMER were further used for a pathway analysis in WebGestalt (Heinz et al., 2010; Wang et al., 2013). Overall design: Nuclei were extracted from skeletal muscles from 10-week-old mice as described in Joshi 2017.