Chromatin Immunoprecipitation for Stem Cell Differentiation

Single ChIP-seq experiment for each H3K4me3, H3K27ac, H3K27ac, and H3K9me3 during differentiation of TSCs into STs and EVTs has been performed following the established procedures^{57 (link)}. In brief, TSCs were fixed with 1% formaldehyde for 7 min at room temperature and then quenched with glycine for 5 min. The fixed cells were sonicated using a Bioruptor (Diagenode) with a 30-s on and 1-min off setting, repeated for 10 min (three cycles). These sheared chromatins were used for immunoprecipitation with 10 µg of native antibodies, including H3K4me3 (Santa Cruz, sc-585), H3K27ac (Santa Cruz, sc-7202X), H3K27me3 (Santa Cruz, sc-9008X), and H3K9me3 (Santa Cruz, sc-8977). The enriched ChIP materials were utilized to generate next-generation sequencing libraries with the NEB ChIP-seq library preparation kit (NEB, E7370L). Subsequently, the ChIP-seq libraries were sequenced using the Illumina NextSeq 500 machine from Illumina.