Genome-Wide Mapping of RORα and RORγt

RORα-TS and RORγt ChIP-Seq was performed as described (Ciofani et al., 2012 (link)) with the following modifications. For each ChIP, 20–80 million cells were cross-linked with paraformaldehyde; chromatin was isolated using truChIP Chromatin Shearing Kit (Covaris) and fragmented with a S220 Focused-ultrasonicator (Covaris). Twin-strep (TS) tagged RORα protein was precipitated using Strep-TactinXT according to the manufacturer’s protocol (IBA Lifesciences). Following immunoprecipitation, the protein-DNA crosslinks were reversed and DNA was purified. DNA from control samples was prepared similarly but without immunoprecipitation. Sequencing libraries were made from the resulting DNA fragments for both ChIP and controls using DNA SMART^™ ChIP-Seq Kit (Takara) for RORα-TS ChIP-Seq and KAPA HyperPlus Kit (Roche) for RORγt ChIP-Seq. The ChIP-Seq libraries were sequenced with paired-end 50 bp reads on an Illumina HiSeq 4000.