Preparation of Mammalian Nascent RNA for Long Read Sequencing

Long read sequencing technologies now allow high-quality sequencing of RNAs (or their cDNAs) that are hundreds to thousands of nucleotides long. Long read sequences of nascent RNA provide single-nucleotide-resolution information about co-transcriptional RNA processing events-e.g., splicing, folding, and base modifications. Here, we describe how to isolate nascent RNA from mammalian cells through subcellular fractionation of chromatin-associated RNA, as well as how to deplete poly(A)⁺ RNA and rRNA, and, finally, how to generate a full-length cDNA library for use on long read sequencing platforms. This approach allows for an understanding of coordinated splicing status across multi-intron transcripts by revealing patterns of splicing or other RNA processing events that cannot be gained from traditional short read RNA sequencing. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Subcellular fractionation Basic Protocol 2: Nascent RNA isolation and adapter ligation Basic Protocol 3: cDNA amplicon preparation.