Transcriptome analysis of S. pombe chromatin reveals cotranscriptional splicing activity. (A) Schematic of nascent RNA (nRNA) purification from chromatin for short- (RNA-seq) and long-read RNA sequencing (LRS). (B) Enrichment of genomic DNA (DNA) and nRNA in the chromatin fraction and depletion of rRNA (18S and 28S) and tRNA revealed by gel electrophoresis and staining with GelStar (Lonza). Western blot analysis with antibodies specific for chromatin-associated proteins Pol II and Histone 3 (H3) and cytoplasmic marker proteins GAPDH and RPL5. (C) Nascent and mRNA-seq read coverage (RPM) over a three-intron gene and a convergent intronless gene. The pooled coverage from three biological replicates for each cellular fraction is shown. To assess splicing levels in nRNA and mRNA, splicing per intron (SPI) was calculated from intron junction reads (Herzel and Neugebauer 2015). SPI values are shown for these representative introns underneath the RNA-seq coverage track. (D) Cumulative SPI distribution for S. pombe introns (nRNA, n = 4481 introns; mRNA, n = 2181). Mean values (line) with standard deviation (shading) are shown for three biological replicates. Gray dashed lines indicate the median splicing levels in the two populations (nRNA 0.59, mRNA 0.95). The inset shows how the SPI is calculated from the number of “spliced” and “unspliced” junction reads spanning a particular intron.
