Chip seq library preparation kit

ChIP reactions were performed as previously^{45 (link)}. Cells were cross-linked with 1% formaldehyde for 7 min at RT, and then formaldehyde was quenched by adding glycine (final 125 mM) for 5 min. After washing cells with PBS two times, fixed cell pellets were resuspended in ChIP dilution buffer and sonicated using a Bioruptor (Diagenode) with a setting of 30 s on and 1 min off for 10 min (3 times). Sheared chromatins containing DNA fragments with an average size of 300 bp were used for IP using 10 μg of a native antibody against each factor. Enriched ChIP samples were used for the generation of sequencing libraries using an NEB ChIP-seq library Preparation Kit (NEB, E6240L) following the manufacturer’s instruction. ChIP-seq libraries were sequenced using Illumina NextSeq 500 machine.

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.