ChIP_Seq analysis in cultured hTSCs

To detect the direct target genes of H4K20me1, H4K20me3, and H3K36me3 in hTSCs, hTSCs are collected and subjected to ChIP-Seq. After aligned to human hg38 by HISAT2, peaks are called by MACS2. Our results show that methylation of H4K20 mediated by PR-SET7 as the key regulator of hTSCs, indicating the essential role of PR-SET7. This ChIP-Seq data provides fundamental information for our further physiological study of PR-SET7. ChIP was performed according to the reported previousl with slight modification. Briefly, about 2x106 TS cells were cross-linked with 1/15 volume of 16% formaldehyde (CST) at room temperature for 10 minutes and quenched with 1/10 volume of 1.25 M glycine for 15 minutes on ice. Cell lysate in lysis buffer III were sonicated using Bioruptor pico (Diagenode) and then incubated with 4 µg antibody overnight at 4℃ with rotation. Immunoprecipitated complexes were collected with 15 µl Protein A Dynabeads (Invitrogen, 10002D) for 1 hour at 4℃ with rotation. Subsequently, beads were washed sequentially once with low-salt buffer, twice with high-salt buffer, once with LiCl buffer, twice with TE, and then eluted in 400 µl of elution buffer for 30 min at 65℃. The eluates were incubation at 65℃ for 8 h to reverse the cross-linking. Next, eluates were treated with proteinase K for 1 h at 55℃ and then RNase A for 30 min at 37℃ before DNA was extracted and purified. The ChIP libraries were prepared using KAPA HyperPrep Kits (Roche, 07962347001) and then run on the Illumina sequencer Hiseq-Xten PE150.