Profiling RNA Polymerase II Phosphorylation Genome-Wide in Fission Yeast.
Kecman T., Heo D-H., Vasiljeva L.
The RNA polymerase II carboxyl-terminal domain (CTD) consists of tandem repeats of consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Dynamic posttranslational modifications of the CTD generate a CTD code crucial for the cotranscriptional recruitment of factors that control transcription, chromatin modification, and RNA processing. Analysis of CTD phosphorylation by ChIP (Chromatin ImmunoPrecipitation) coupled with high-throughput DNA sequencing (ChIP-seq) is a powerful tool to investigate the changes in CTD phosphorylation during the transcription cycle. In this chapter, we describe a ChIP-seq protocol to profile the different CTD phospho-marks in fission yeast. Using this protocol, we have found that Tyr1P, Ser2P, and Thr4P signals are highest at gene 3' ends, whereas Ser5P is enriched across the gene bodies.