GSE196222
ChIP_Seq analysis in cultured mTSC cells with or without Dox
To detect the direct target genes of H4K20me1, H4K20me3, and H3K4me3  in mTSCs, mTSCs with or without Dox are collected and subjected to ChIP-Seq. After aligned to mouse mm10 by HISAT2, peaks are called by MACS2. Our results show that methylation of H4K20 mediated by Pr-set7 as the key regulator of mTSCs, indicating the essential role of Pr-set7. This ChIP-Seq data provides fundamental information for our further physiological study of Pr-set7. Overall design: 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.