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. 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.

为检测人类滋养层干细胞（hTSCs）中H4K20单甲基化（H4K20me1）、H4K20三甲基化（H4K20me3）以及H3K36三甲基化（H3K36me3）的直接靶基因，本研究收集hTSCs并开展染色质免疫共沉淀测序（ChIP-Seq）实验。测序reads通过HISAT2比对至人类参考基因组hg38后，采用MACS2进行峰位点识别。本研究结果显示，由PR-SET7介导的H4K20甲基化是hTSCs的关键调控因子，证实了PR-SET7发挥着不可或缺的重要作用。本次ChIP-Seq数据集为后续开展PR-SET7的生理学研究提供了基础数据支撑。

实验整体设计：染色质免疫共沉淀实验参考已发表方案并稍作修改。简要步骤如下：取约2×10^6个TS细胞，于室温下用1/15体积的16%甲醛（CST公司产品）交联10分钟，随后加入1/10体积的1.25 M甘氨酸于冰上淬灭反应15分钟。将细胞重悬于裂解缓冲液III中，使用Bioruptor Pico超声破碎仪（Diagenode公司）进行超声处理，随后加入4 μg抗体，于4℃旋转孵育过夜。使用15 μl Protein A磁珠（Invitrogen，货号10002D）于4℃旋转孵育1小时，收集免疫沉淀复合物。随后，磁珠依次经低盐缓冲液洗涤1次、高盐缓冲液洗涤2次、氯化锂（LiCl）缓冲液洗涤1次、TE缓冲液洗涤2次，之后用400 μl洗脱缓冲液于65℃洗脱30分钟。将洗脱液置于65℃孵育8小时以逆转交联反应。随后，洗脱液依次经55℃蛋白酶K处理1小时、37℃核糖核酸酶A（RNase A）处理30分钟，之后进行DNA提取与纯化。使用KAPA HyperPrep试剂盒（Roche，货号07962347001）构建ChIP测序文库，随后在Illumina HiSeq-Xten PE150测序平台完成测序。