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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