Impact of CTD truncation on co-transcriptional splicing.

For overexpression of exogenous RPB1, 10 ug of purified HA-tagged RPB1 plasmids (wildtype or CTD-truncated, both α-amanitin resistant) were transfected into HeLa S3 cells per 10 cm dish using TurboFect transfection reagent (Thermo Fisher Scientific). Culture medium was changed to include 2 ug/ml of Doxycycline 12 hrs post-transfection for inducing exogenous gene expression and then treated with 2.5 ug/ml α-amanitin to block the function of endogenous RPB1. Cells were further cultured for 42 hrs before harvesting for subsequent analysis. We expressed a-amanitin resistant full-length and CTD truncated RBP1 and compared their impact on co-transcriptional splicing after inhibiting the endogenous Pol II in HeLa cells.