LINE1 and PRC2 control nucleolar organization and repression of the 8C-state in human ESCs [RNA-Seq_RSeT+DT_H9_L1KD+siRNA-KD]

To study siRNA-mediated TPRX1, TP53 and H3.XY co-knockdown effected when transfected together with L1 targeting anti sense oligos (ASO-L1) in RSeT+DT naïve hES cells. Overall design: In this study, we investigated if co-knockdown of TPRX1, TP53 or H3.XY expression would reverse the ASO-L1 knockdown induced 8-cell (8C) stage-specific genes upregulation. RSeT media maintained naive H9 hES cells were transfected with non-specific targeting control anti-sense oligos (ASO-Ctr) or ASO-L1, together with 6-FAM (Fluorescein) labeled siRNAs targeting non-specific sequence (NTsi), TPRX1 (TPRX1si), TP53 (TP53si) and H3.XY (H3.XYsi), respectively. Transfected cells were maintained in the RSeT media added with 10 nM DZNep and 5nM TSA (RSeT+DT). 48 hours later, FAM+ cells were FACS collected and immediately processed for RNA extraction and RNA-seq library prep.

本研究旨在探究在RSeT+DT培养基培养的初始态人胚胎干细胞（naïve hES cells）中，与靶向L1的反义寡核苷酸（ASO-L1）共转染时，由小干扰RNA（siRNA）介导的TPRX1、TP53及H3.XY联合敲低的效应。整体实验设计：本研究旨在验证TPRX1、TP53或H3.XY的联合敲低是否能够逆转ASO-L1敲低所诱导的8细胞阶段特异性基因上调现象。将在RSeT培养基中维持培养的初始态H9人胚胎干细胞，分别转染靶向非特异性序列的对照反义寡核苷酸（ASO-Ctr）或ASO-L1，同时辅以6-羧基荧光素（6-FAM，Fluorescein）标记的靶向非特异性序列的siRNA（NTsi）、靶向TPRX1的siRNA（TPRX1si）、靶向TP53的siRNA（TP53si）及靶向H3.XY的siRNA（H3.XYsi）。转染后的细胞在添加了10 nM DZNep与5 nM曲古抑菌素A（TSA）的RSeT培养基（即RSeT+DT培养基）中继续培养。转染48小时后，通过荧光激活细胞分选（FACS）收集6-FAM阳性细胞，并立即进行RNA提取与RNA测序（RNA-seq）文库构建。