Lucas, Morghan C.Pryszcz, Leszek Piotr, 1985-Medina, RebecaMilenkovic, IvanCamacho, NoeliaMarchand, VirginieMotorin, YuriRibas de Pouplana, LluísNovoa, Eva Maria2023-06-192023-06-192024Lucas MC, Pryszcz LP, Medina R, Milenkovic I, Camacho N, Marchand V, et al. Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing. Nat Biotechnol. 2024 Jan;42(1):72-86. DOI: 10.1038/s41587-023-01743-61087-0156http://hdl.handle.net/10230/57222Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress.application/pdfeng© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.info:eu-repo/semantics/articlehttp://dx.doi.org/10.1038/s41587-023-01743-6RNA sequencingSmall RNAsinfo:eu-repo/semantics/openAccess