Minelute spin columns kit

Chromatin immunoprecipitation was performed by standard procedures, as previously described [55], followed by sonication with a Covaris S220 Ultra-Sonicator system. Chromatin DNA was purified using the MinElute Spin Columns Kit (Qiagen). ChIP-seq library preparation and sequencing were performed by the McGill University Genome Quebec Innovation Centre with Illumina HiSeq 2000 as single-end 50 nucleotide reads, according to Illumina instructions with input chromatin DNA used as control. Sequencing data was mapped to the mm9 assembly of the mouse genome with BWA-MEM [56], marking shorter split hits as secondary, and processed for visualization of ChIP-seq signals, as previously described [14]. Peak detection was performed using the MACS software [57], whereas de novo motif discovery was completed with Homer [58] on the 100 base pairs surrounding the center of the peaks. Homer was employed to classify overlapping peaks that were defined as being co-localized within 100 bp of one another. Peak annotation followed by gene ontology (GO) was performed by GREAT v3.0.0 [59], utilizing the whole genome as background and the single nearest gene association rule.

Cells were crosslinked and sonicated followed by chromatin immunoprecipitation as previously described (29 (link)). Antibodies against H3K9ac, H3K18ac, H3K27ac, H4K8ac and H3K27me3 were obtained from Abcam (ab4441, ab1191, ab4729, ab15823 and ab6002), RXRα, and p300 from Santa Cruz (sc-553x and sc-32758x). DNA was purified using the MinElute Spin Columns Kit (Qiagen) and input chromatin DNA was used as control. Purified DNA was sequenced by the McGill University Genome Quebec Innovation Centre with Illumina HiSeq 2000. Sequencing reads were mapped to the mouse genome build mm9 using Bowtie, allowing for three mismatches and reporting the single best alignment per 50 bp read. Picard was used to filter out replicated reads (http://picard.sourceforge.net/), and BAM files were converted into BED files with the BEDTools suite (33 (link)). For visualization of ChIP-seq signals in the Integrative Genomics Viewer, aligned reads were extended by 125 bp at their 3′ end and basewise signal intensity was computed. Local peaks in read enrichment were identified using MACS (34 (link)) (v1.0.0) with a P-value threshold of 1 × 10⁻⁵.