We also visually inspected the locations of several of the LRH-1 binding sites with respect to neighboring FXR binding peaks, using peak distribution tracks in the UCSC genome browser.
14 We characterized the genomic location of binding peaks using a peak-finding tool of Avadis NGS that classifies the locations into the upstream region, 5′ untranslated region (5′UTR), exon, intron, and 3′UTR.
For each ChIP-seq dataset, we predicted the location of a CTCF binding site in each peak using the GADEM software [ 56].
We also inspected LRH-1 binding peaks by using the bedGraph format that allows a display of continuous-valued ChIP-seq data in track format using the UCSC genome browser.
The ChIP-Seq binding peaks were scored using the SPP peak caller and irreproducible discovery rate analysis (Kharchenko et al. 2008; Li et al. 2011), which resulted in the identification of 1000−6500 strongly reproducible peaks per cell line (Table 1).
Dillon and colleagues found 261 binding sites using the ChIPOTle peak finding program.
Accordingly, we sequenced two independent ChIP-seq replicates and determined a set of enriched ZNF217 binding sites, using the Sole-Search peak calling tool [ 12], by taking the shared ZNF217 binding sites (False Discovery Rate (FDR) < 0.0001) from both replicates (18, 965 binding sites; Additional file 1).
Unambiguously mapped and unique reads were kept for subsequent generation of binding profiles and calling of peaks using MACS with an fdr < 0.05 [ 61].
Analyzed uniquely mapped reads used for analysis (Bold) To identify LRH-1 binding peaks, we used Model-based Analysis of ChIP-seq (MACS), which was designed to analyze data generated by short read sequencers such as from the SOLiD platform [ 15] to first estimate peak size and location, using BED files as an input.
We also analyzed individual peaks for differential binding across conditions using the EdgeR tool [ 30], but were only able to identify 12 regions (0.2% of total) with significant differential CREB enrichment at a 10% FDR threshold (Additional file 1: Table S2).
