Synthesis of modified nucleotide polymers by the poly(U) polymerase Cid1: application to direct RNA sequencing on nanopores

Capture and analysis of RNA polymerase II (RNAPII) associated nascent transcripts by Cid1 I-tailing. (A) Protocol for isolating RNAPII-associated transcripts from chromatin (see Materials and Methods). (B) A model for cotranscriptional splicing. Introns bounded by 5′ splice sites (green) and 3′ splice sites (gold) are removed creating exon–exon junctions (blue) in nascent transcripts as RNAPII (magenta) moves down the gene. (C) Genome browser views of nanopore reads mapping to ACT1 (top), RPS4B (middle), and FBA1 (bottom). The 3′ ends of reads from nascent RNAs are distributed along the gene, and splicing usually occurs before RNAPII has moved 100 nt beyond the 3′ splice site.