Exploring Extracellular Vesicle Transcriptomic Diversity Through Long-Read Nanopore Sequencing.

Nanopore long-read RNA sequencing is reshaping extracellular vesicle (EV) research by providing the capacity to analyze full-length RNA molecules. EVs are crucial for intercellular communication, carrying a diverse range of RNA cargo that can regulate recipient cell behavior. However, traditional short-read sequencing methods involve transcript fragmentation, limiting our understanding of the EV transcriptomic landscape. Furthermore, it has been generally assumed that EV RNAs are likely to be fragmentation products of cellular RNAs, and the extent to which full length RNAs are present within EVs remains to be clarified. Recent advancements in sequencing technology, particularly long-read sequencing by Oxford Nanopore Technologies (ONT), offer a solution to this limitation. Hence, long-read sequencing allows for the analysis of full-length EV RNA molecules, providing deeper insights into their integrity and isoform diversity. Here, we present a comprehensive protocol for EV RNA purification, cDNA library preparation, and sequencing using ONT's MinION platform.