Profiling the polyadenylated transcriptome of extracellular vesicles with long-read nanopore sequencing.

BACKGROUND: While numerous studies have described the transcriptomes of extracellular vesicles (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-targeted RNA populations. It has been assumed that mRNA signatures in EVs are likely to be fragmentation products of the cellular mRNA material, and the extent to which full-length mRNAs are present within EVs remains to be clarified. RESULTS: Using long-read nanopore 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 443 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 10.58% of all EV reads, and 18.67% of all cellular (WC) reads, corresponded to known full-length transcripts, with mRNAs representing the largest biotype for each group (EV = 58.13%, WC = 43.93%). 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. CONCLUSION: This work provides novel insights into the compositional diversity of poly-A transcript isoforms enriched within EVs, while also underscoring the potential usefulness of nanopore sequencing to interrogate secreted RNA transcriptomes.

RNA Interference (RNAi) Screening in Cultured Drosophila Cells.

Genetic perturbation assays have been crucial to the discovery of molecular pathways that drive diverse biological processes. RNA interference (RNAi)-mediated depletion of gene products represents a powerful means of elucidating gene function, as it allows one to systematically probe the phenotypic effects resulting from the functional loss of specific targets. The relative ease of use of RNAi technologies in cultured cells has allowed the design and implementation of genome-wide investigations to systematically reveal gene function. In this chapter, we describe methods for high-throughput RNAi-mediated loss-of-function studies in cultured cells of Drosophila melanogaster. First, we describe the in vitro synthesis of double stranded RNAs (dsRNAs) from a genome-wide Drosophila RNAi library. Next, we outline the procedures used to carry out high-throughput RNAi screens using a cell bathing approach and high-content screening microscopy, illustrating how these experiments can be utilized to study specific cellular contexts, such as cellular stress. Finally, we illustrate some approaches commonly employed to validate the depletion of identified gene candidates.

Draft Genome Sequence of Aeromonas caviae Strain 429865 INP, Isolated from a Mexican Patient.

Aeromonas caviae is an emerging human pathogen. Here, we report the draft genome sequence of Aeromonas caviae strain 429865 INP which shows the presence of various putative virulence-related genes.