ChIP-seq Protocol with Modifications

ChIP was carried out as described before with some modifications (63 (link), 64 (link)). Briefly, cells were cross-linked by adding 1% (w/v) formaldehyde and incubated for 20 min at RT with gentle shaking. Cell fixation was interrupted by adding 110 mM glycine. Cells were scraped off and resuspended in lysis buffer following the iDeal ChIP-qPCR kit protocol (Diagenode, Liège, Belgium). To obtain genomic DNA fragments between 500 and 100 bp, cell lysates were sonicated for four rounds of ten cycles (30 s ON/30 s OFF) using the Bioruptor Pico (Diagenode) at high power setting. For immunoprecipitations, the following antibodies were used: 1 μg of rabbit polyclonal anti-IgG (C15410206, Diagenode) as negative control IP; 1 μg of mouse monoclonal anti-FOXJ1 (14-9965-82, Thermo Fisher Scientific) and 3.8 μg of mouse monoclonal anti-FLAG (F1804, Sigma Aldrich). Chromatin–antibody complexes were immunoprecipitated by DiaMag Protein A-coated magnetic beads (Diagenode). DNA isolation and de-cross-linking was carried out as described by the iDeal ChIP-qPCR kit protocol (Diagenode). Coprecipitated DNA was quantified by real-time qPCR using the primers listed in Table S2.