Long-read Sequencing of Nascent RNA from Budding and Fission Yeasts.

Gene expression requires DNA transcription and simultaneous RNA processing steps that transform the precursor RNA into fully mature RNA. In eukaryotes, the processing of protein-encoding messenger RNAs (mRNAs) includes 5' end capping, editing, splicing, RNA modification, poly-adenylation cleavage, and polyadenylation. Short-read sequencing of total or messenger RNA largely reveals the final output of transcription and processing because it utilizes 1) steady-state, mature RNA that is mostly processed and 2) sequencing reads that are too short to detect adjacent processing events (e.g. two adjacent introns). In contrast, long-read sequencing of nascent RNA allows the detection of rarer, full-length transcripts that are in the process of being transcribed and processed. The 3' end of each nascent RNA establishes the position of RNA polymerase II (Pol II) along the gene at the time of cell lysis, providing a 'timeline' for RNA processing events. In addition, the density of 3' ends along genes or at gene landmarks reflects Pol II density, which is related to changes in transcription elongation rate. In organisms with complex gene architectures, information about splicing across multiple introns within the same transcript can be extracted, as well as the location of transcription start sites (TSSs) and polyA cleavage sites. Here, we describe the isolation of nascent RNA from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, preparation of a cDNA library for long-read sequencing on Oxford Nanopore Technologies or Pacific Biosciences platforms, and initial data analysis steps. These methods comprise versatile and powerful tools for the investigation of coupled RNA synthesis and processing.