Rpb9 plays a critical role in global co-transcriptional splicing (Fraction RNA-seq)

In order to measure the impact of Rpb9 on co-transcriptional splicing, we treated HeLa cells with control siRNA or siRpb9, and for comparative analysis, we also depleted Rpb1 and confirmed by Western blotting the knockdown efficiency in all cases. To measure co-transcriptional splicing, we fractionated HeLa cells into cytosolic (Cyt), nucleoplasm (NP), and chromatin/particle (Chr) fractions. We purified total RNA from each compartment, spiked in with in vitro transcribed EGFP and FireFly luciferase RNA for data normalization, depleted ribosomal RNAs, and constructed two independent libraries for RNA-seq, which showed excellent reproducibility . Overall design: Characterization of co-transcriptional splcing after Rpb9 knock-down in Hela cells