ChIP was conducted as previously described (Komashko et al., 2008 (link)). H460 cells were fixed in 1% formaldehyde (Sigma) for 15 minutes at 25 °C. After lysis, samples were sonicated using a biorupter sonicator (Diagenode) for 60 cycles (30 seconds per cycle/30 seconds cooling) at a high power level. Chromatin sheering was optimized to a size range of 200 to 600 bp. Chromatin (100 μg) was immunoprecipitated with the NR0B1 antibody (Cell Signaling Technology). For DNA sequencing, samples were prepared for library construction, flow cell preparation and sequencing were performed according to Illumina’s protocols. Sequencing was accomplished on Illumina HiSeq 2500 using PE 2 × 125 bp reads with over 14 million clusters per sample.
Sequencing reads were aligned to the hg19 genome using bowtie2 (Langmead and Salzberg, 2012 (link)). Peak detection was carried out using HOMER (homer.ucsd.edu), comparing the NR0B1 IP sample against a whole-cell extract (WCE) with default parameters for transcription factor-style analysis. This requires relevant peaks to be significantly enriched over WCE and the local region with an uncorrected Poisson distribution-based p-value threshold of 0.0001 and false discovery rate threshold of 0.001. These peaks were further restricted to a 2kb window around annotated transcription start sites.
Sequencing reads were aligned to the hg19 genome using bowtie2 (Langmead and Salzberg, 2012 (link)). Peak detection was carried out using HOMER (homer.ucsd.edu), comparing the NR0B1 IP sample against a whole-cell extract (WCE) with default parameters for transcription factor-style analysis. This requires relevant peaks to be significantly enriched over WCE and the local region with an uncorrected Poisson distribution-based p-value threshold of 0.0001 and false discovery rate threshold of 0.001. These peaks were further restricted to a 2kb window around annotated transcription start sites.