Nebnext chip seq library prep reagent

High-throughput sequence libraries were generated from immunoprecipitated and IgG eluted DNA using the NEBNext ChIP-seq library prep reagent set for Illumina sequencing, according to the manufacturer's instructions. Fifty-cycle paired-end sequencing was performed by the University of Wisconsin–Madison Biotechnology Center. Image processing and sequence extraction were performed using the standard Illumina Pipeline. Raw paired-end sequencing files were assessed for quality using FastQC (https://www.bioinformatics.babraham.ac.uk/), trimmed using the Trim Galore! package in R (https://github.com/FelixKrueger/TrimGalore), and aligned to the mm9 genome using Bowtie 2 v2.3.4 (Langmead et al. 2009 (link)), local alignment. Following alignment, SAMtools to filter multimapping reads (Li et al. 2009 (link)) and PCR duplicates. Uniquely mapped and filtered sequence reads were mapped to DhMRs found to contain a putative CLOCK binding site (CACGTG) by motif enrichment analysis (N = 577). Only data from these sites were used for differential binding analyses (R package edgeR) (Robinson et al. 2010 (link)), after controlling for background through normalization to sequenced IgG reads. A P-value threshold of 0.05 was used to determine significant differential binding of CLOCK between groups.