ChIP-seq Protocol for Transcription Factors

ChIP experiments were conducted as previously described (23 (link)). Briefly, TSCs were fixed with 1% formaldehyde for 7 min at room temperature, and then glycine (final 125 mM) was added to quench formaldehyde (5 min). The fixed cells were sonicated using a Bioruptor (Diagenode) with a setting of 30 s on and 1 min off for 10 min (total of three times), and the sheared chromatins that have an average of 250 bp DNA fragments were utilized for immunoprecipitation using 10 μg of a native antibody. The antibodies used include EP300 (Santa Cruz, sc-585), FOS (Santa Cruz, sc-7202X), GATA2 (Santa Cruz, sc-9008X), MAFK (Santa Cruz, sc-477X), TEAD4 (Abcam, ab58310), TFAP2C (Santa Cruz, sc-8977) and H3K27ac (Active Motif, 39133). Enriched ChIP materials were used to generate next-generation sequencing libraries using an NEB ChIP-seq library preparation kit (NEB, E7370L). ChIP-seq libraries were sequenced using an Illumina HiSeq 2500 machine.