Investigating RNA dynamics from single molecule transcriptomes

Investigating RNA dynamics is crucial for uncovering fundamental mechanisms, such as alternative splicing, RNA stability, and post-transcriptional modifications, all processes with implications for identifying therapeutic targets and advancing knowledge of cellular function and regulation. Advances in long-read sequencing technologies, particularly from Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), offer unprecedented insights into RNA dynamics at single molecule and single nucleotide resolutions. In this review, we examine protocols and methods for analyzing RNA dynamics, focusing on isoform detection, poly(A) tail length quantification, and mapping of RNA modifications. We envision that these high-throughput, transcriptome-wide data sets, combined with integrated software systems, will transform workflows for studying single molecule RNA dynamics. Such advances will help unravel the complexities of gene regulation and deepen our understanding of cellular processes.