LINE1 and PRC2 control nucleolar organization and repression of the 8C-state in human ESCs [ChIRP-seq,RSeT+DT H9]

In this study, we profiled for LINE1 binding loci in RSeT+DT naïve hES cells with the Chromatin Isolation by RNA Purification (ChIRP)-seq strategy. We followed a previously published ChIRP protocol described (Percharde et al. 2018; Lu et al. 2020). RSeT H9 hES cells were witched to RSeT media added with 10 nM DZNep and 5nM TSA (RSeT+DT) for 48 hours, then collected for preparing ChIRP sequencing sample. x107 cells per ChIRP pulldown were subjected to DNAseI digestion after crosslinking the cells. After brief sonication and spinning, chromatin supernatants were collected for hybridization with probes. After 3 hour of hybridization with LINE1 probes or the control MALAT1 probes, streptavidin M280 beads were added and incubated for another 3 hours. After hybridization, the beads were washed 5 times and then treated with RNase H to elute ChIRP pulled down DNAs, which were reverse crosslinked and purified, and then used for library preparation. More protocol details are described in the methods of this study.

本研究采用RNA纯化染色质分离测序（Chromatin Isolation by RNA Purification-seq, ChIRP-seq）技术，对RSeT+DT处理的初始态人类胚胎干细胞（naïve hES cells）中的长散布核元件1（LINE1）结合位点进行了全基因组图谱分析。本研究沿用已发表的ChIRP实验方案（Percharde等，2018；Lu等，2020）。将RSeT培养基培养的H9初始态人类胚胎干细胞转移至添加了10 nM DZNep与5 nM TSA的RSeT培养基（即RSeT+DT组）中培养48小时，随后收集细胞用于制备ChIRP测序样本。每轮ChIRP亲和纯化下拉实验使用1×10^7个细胞，在细胞交联后对其进行脱氧核糖核酸酶I（DNase I）消化。经短暂超声破碎与离心后，收集染色质上清液用于与探针杂交。与LINE1探针或对照肺腺癌转移相关转录本1（MALAT1）探针杂交3小时后，加入链霉亲和素M280磁珠并继续孵育3小时。杂交完成后，将磁珠洗涤5次，随后用核糖核酸酶H（RNase H）处理以洗脱ChIRP亲和纯化得到的DNA；洗脱所得DNA经反交联与纯化后，用于测序文库构建。本研究的方法部分详述了更多实验流程细节。