Ssu72 and Ser5P-RNAPII ChIPseq in hESCs. GROseq analysis in siCtrl and siSSu72 transfected hESCs

Ssu72 is a Ser5P-RNAPII phosphatase, nevertheless few targets have been characterized and its role in mammal cells remains elusive. Using ChIPseq and GROseq technologies we aimed to identify Ssu72 targets in human embryonic stem cells. For the ChIPseq experiments, H1 hESCs growing in mTeSR media were doubled fixed with DSG 2mM (45min) and FA 1% (20min) and processed to chromatin immunoprecipitation. For GROseq experiments, H1 hESCs were transfected with siCtrl or siSsu72 siRNAs using RNAimax (Invitrogene) for 48h. Then, run-on experiment was done with isolated nuclei. Nascent RNA purification and subsequent steps needed before sequencing were carried out essentially as described in Core et al, 2008, Science 319(5871): 1791-1792.