Profiling the Full-Length Transcriptome of Extracellular Vesicles With Oxford Nanopore Sequencing

While numerous studies have described the transcriptomes of EVs in different cellular contexts, these efforts have typically relied on sequencing methods requiring RNA fragmentation, which limits interpretations on the integrity and isoform diversity of EV-encapsulated RNA populations. Furthermore, it has been assumed that mRNA signatures in EVs are likely to be fragmentation products of the cellular mRNA material, and little is known about the extent to which full-length mRNAs are present within EVs. Using Oxford nanopore long-read RNA sequencing, we sought to characterize the full-length polyadenylated (poly-A) transcriptome of EVs released by human chronic myelogenous leukemia K562 cells. We detected 441 and 280 RNAs that were respectively enriched or depleted in EVs. EV-enriched poly-A transcripts consist of a variety of biotypes, including mRNAs, long non-coding RNAs, and pseudogenes. Our analysis revealed that 12.72% of all reads present in EVs corresponded to known full-length transcripts, 65.34% of which were mRNAs. We also observed that for many well-represented coding and non-coding genes, diverse full-length transcript isoforms were present in EV specimens, and these isoforms were reflective-of but often in different ratio compared to cellular samples. Here we report a full-length transcriptome from human EVs, as determined by long-read nanopore sequencing. Overall design: Total RNA from K-562 cells and EVs was isolated and the sequencing libraries were prepared using Nanopore PCR-cDNA Barcoding Kit.