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

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. Overall design: 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.