title: GSM9211240: Input DNA for REST KO; Mus musculus; ChIP-Seq
library_name: GSM9211240
library_construction_protocol: Uteri from Restf/f and Restf/f PRCre/+ mice (3 of each genotype, in diestrus) at 6 months of age were minced on ice and cross-linked using 1% formaldehyde. Chromatin IP for PGR and REST were performed according to published protocols (Varghese et al., PNAS, 2010. Rubel et al., Mol Endocrinol, 2012.) An unbiased whole genome profiling of REST and PGR binding was obtained by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq). ChIP-Seq data was generated from REST-ChIP in control (Restfl/fl), REST-ChIP in Rest conditional knockout (Restfl/fl PR+/Cre), PR-ChIP in control (Restfl/fl), and PR-ChIP in Rest conditional knockout (Restfl/fl PR+/Cre) samples. We used corresponding input DNA controls from WT and KO samples to measure total chromatin before IP. ChIP-Sequencing was performed in an Illumina NovaSeq 6000 sequencing machine (Illumina, San Diego, CA) at a 100-base paired-end read resolution. After read quality assessment (FastQC [Andrews 2010]) and adapter trimming (Cutadapt [Martin, EMBnet.journal, 2011 ]), the sequenced reads were mapped to the mouse reference genome (GRCm38) using the Bowtie2 [Langmead et al., Nat Methods, 2012] software. Sequencing produced between 31.5 and 56.5 million reads per sample of which 97% to 99% mapped to the reference genome. Significantly enriched ChIP regions (relative to the corresponding Input sample) were detected using the Model-based Analysis of ChIP-Seq (MACS2 [Zang et al., Genome Biol, 2008]) software. The analysis was run under default settings in the paired-end mode with the q-value cutoff for calling significant regions set at 0.05. The peak regions were scanned for RE-1 and Pgre binding motifs with a position weight matrix (PWM) similarity cutoff set at 70%. The number of significantly enriched regions (enrichment FC ≥ 1.5, q-value ≤ 0.05) detected for each antibody is given in Figure 4C. The mapped regions were visualized using the Integrative Genome Viewer (IGV [Thorvaldsdottir et al., Brief Bioform, 2013]). A motif analysis for known transcription factor binding sites in the enriched regions was performed using the HOMER [Heinz et al., Mol Cell, 2010] software. The heatmaps of sequencing-depth-normalized read coverage around 3 kb up-stream and down-stream from the transcription start site (TSS) of all genes were generated using the deepTools [Ramirez et al., Nucleic Acids Res, 2014] software.