Nascent transript profiling upon in mouse embryonic stem cells under control conditions or Phf5a knockdown

Phf5a regulates transcription elongation in mouse embryonic stem cells (ESCs), through regulation of the Paf1 complex. In this study we assayed nascent RNA profiling using global run-on sequncing in mouse ESCs under conditions of self-renewal, differentiation and Phf5a knockdown. These results revealed that genes positively regulated by Paf1 in ESCs exibit RNA elongation pausing on their promoters. We used conditions of self-renewal, differentiation, Phf5a knockdown and Paf1 knockdown to monitor nascent transcript expression changes during Phf5a depletion using global run-on RNA sequencing (GRO-seq). These results revealed that Phf5a through the control of Paf1 complex regulates pause release and elongation of pluripoency-associated genes in embryonic stem cells. Nascent RNA was extracted from 10 million self-renewing, differentiated or Phf5a knockdown treated ESCs using Trizol extraction (Invotrogen) according to the manufacturer’s protocol following the global run-on (GRO) reaction and BrUTP labeling. Nascent BrUTP-containing RNA was purified using BrdU beads (Santa Cruz) which was then used for library preparation. Libraries were sequenced on the Illumina HiSeq 2000 using 50bp single-read method. Pause release analysis was performed for each matched shControl versus shPhf5a knockdown in each biological replicate.

Phf5a通过调控PAF1复合物（Paf1 complex），参与小鼠胚胎干细胞（ESCs）的转录延伸过程。本研究针对小鼠胚胎干细胞（ESCs）在自我更新、分化以及Phf5a敲低三种培养条件下的样本，采用全局连缀测序（global run-on sequencing，GRO-seq）技术检测新生RNA表达谱。结果显示，在ESCs中受Paf1正向调控的基因，其启动子区域存在RNA延伸暂停现象。本研究同时设置自我更新、分化、Phf5a敲低以及PAF1敲低四种培养条件，借助全局连缀RNA测序（GRO-seq）技术，监测Phf5a缺失过程中新生转录本的表达变化。结果表明，Phf5a通过调控PAF1复合物，可调控胚胎干细胞中多能性相关基因的暂停释放与延伸过程。在完成全局连缀（GRO）反应与BrUTP标记后，本研究从10^7份经自我更新培养、分化处理或Phf5a敲低处理的ESCs中提取新生RNA，操作严格按照制造商提供的实验方案进行Trizol提取（Invotrogen）。随后通过BrdU磁珠（Santa Cruz）纯化含有BrUTP的新生RNA，所得产物用于文库构建。文库在Illumina HiSeq 2000测序平台上采用50bp单端读长策略进行测序。针对每一次生物学重复中匹配的shControl与shPhf5a敲低样本，均开展了暂停释放分析。