Rpb9 plays a critical role in global co-transcriptional splicing in mouse ESC (Fraction chrRNA-seq)

To define the function of Rpb9 on co-transcriptional splicing, we used auxin-inducible Rpb9 degron system in mouse embryonic stem cells (mESCs) with auxin treatment for 3 hours to sufficiently deplete Rpb9. To measure co-transcriptional splicing, we fractionated mESCs cells (spiked in with Drosophila S2 cells for data normalization) into cytosolic (Cyt), nucleoplasm (NP), and chromatin/particle (Chr) fractions. We purified total RNA from Chr compartment, depleted ribosomal RNAs, and constructed two independent libraries for RNA-seq, which showed excellent reproducibility. Characterization of co-transcriptional splicing after degradating Rpb9 in mESC cells