H2AK119ub1 native ChIP-seq upon Xist indution

Native ChIP-seq for H2AK119ub1 upon Xist induction in iXist-ChrX mouse embryonic stem cells. Comparison of WT with Spen RRM deletion and SPOC domain mutants for two timepoints of Xist induction, 3h and 24h. Overall design: ChIP-seq for H2AK119ub1 was performed on native chromatin extract from WT and biological replicate clones for Spen RRM deletion and SPOC mutation cell lines derived from parental iXist-ChrX cells. For the purposes of this report H2AK119ub1 distribution acts a surrogate marker for Xist RNA distribution across the Xi chromosome, and its pattern is clearly abnormal in Spen RRM deletion but not SPOC mutant cells. Two time points of Xist induction, 3h and 24h were profiled for comparison. Whereas all samples for the 3h experiment were processed in parallel on the same day, for the 24h experiment the Spen RRM deletion lines, SPOC mutant lines, and each of the three WT replicates were processed on separate occasions. For 24h data sets (WT Reps1&2, SpenRRM, SPOCmut), Drosophila SG4 cells were carefully added as a spike in (40 million mES cells with 10 million SG4 cells) upon collection and prior to chromatin extraction. Although spike-in normalisation was not performed for this study, input samples are provided here for this analysis.

针对iXist-ChrX小鼠胚胎干细胞中X失活特异性转录本（Xist）诱导后组蛋白H2A赖氨酸119位单泛素化（H2AK119ub1）的原生染色质免疫共沉淀测序（Native ChIP-seq）实验。本研究针对Xist诱导的两个时间节点（3小时与24小时），比较了野生型（WT）、Spen蛋白RRM结构域缺失细胞系以及SPOC结构域突变细胞系的测序结果。
整体实验设计：针对H2AK119ub1的Native ChIP-seq实验，取材自亲本iXist-ChrX细胞衍生的野生型细胞、Spen RRM缺失细胞系以及SPOC结构域突变细胞系的生物学重复克隆的原生染色质提取物。本报告中，H2AK119ub1的分布可作为Xist RNA在失活X染色体（Xi）上分布的替代标记物；其分布模式在Spen RRM缺失细胞中存在显著异常，但在SPOC结构域突变细胞中并无此现象。本研究选取Xist诱导的3小时与24小时两个时间点进行测序分析以作比较。3小时时间点的所有样本均于同日同步完成实验处理，而24小时时间点的Spen RRM缺失细胞系、SPOC结构域突变细胞系以及三组野生型生物学重复样本则分别独立完成实验处理。针对24小时时间点的数据集（野生型重复1、2，SpenRRM突变体及SPOCmut突变体），在样本收集后、染色质提取前，均按4000万小鼠胚胎干细胞（mES）搭配1000万果蝇SG4细胞的比例，精准加入果蝇SG4细胞作为外参对照（spike-in）。尽管本研究未进行外参归一化分析，但本次分析已附带提供了输入对照样本。