Identification of regulatory links between transcription and RNA processing with long-read sequencing

Summary: We present a detailed protocol for sequencing full-length mRNA isoforms using the Oxford nanopore long-read sequencing technology. We describe steps for poly(A) RNA isolation, library preparation, and cDNA size selection. We then detail procedures for sequencing and processing and a computational framework to identify exon couplings and assign mRNA 5′ ends and 3′ ends to each other. Our approach enables the identification of links between transcription initiation and co-transcriptional RNA processing events.For complete details on the use and execution of this protocol, please refer to Alfonso-Gonzalez et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.