Characterization of RNA Polymerase II occupancy 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 for genome-wide localization of Ser-5-phosphorylated RNA polymerase II and Ser-2-phosphorylated RNA polymerase II in mouse ESCs under conditions of shControl and shPhf5a knockdown. These results revealed that downregualtion of Phf5a results in the increase of the initiating form of RNA polymerase II (Ser5-phosphorylated) and in the aberrant loss of the elongating form of RNA polymerase II (Ser2-phosphorylated) of pluripotency genes in ESCs. Ten million cells were used for the ChIP and precipitated using 10 micrograms of antibody against Ser-5-Phosphoryalted RNA polymerase II (Abcam, ab5131 ) and against Ser-2-Phosphoryalted RNA polymerase II (Active Motif, clone 3E10)