Salmen, FredrikDe Jonghe, JoachimKaminski, Tomasz S.Alemany, AnnaParada, Guillermo E.Verity-Legg, JoeYanagida, AyakaKohler, Timo N.Battich, Nicholasvan den Brekel, FlorisEllermann, Anna L.Martínez Arias, AlfonsoNichols, JenniferHemberg, MartinHollfelder, Florianvan Oudenaarden, Alexander2022-09-082022-09-082022Salmen F, De Jonghe J, Kaminski TS, Alemany A, Parada GE, Verity-Legg J, Yanagida A, Kohler TN, Battich N, van den Brekel F, Ellermann AL, Arias AM, Nichols J, Hemberg M, Hollfelder F, van Oudenaarden A. High-throughput total RNA sequencing in single cells using VASA-seq. Nat Biotechnol. 2022 Jun 27. DOI: 10.1038/s41587-022-01361-81087-0156http://hdl.handle.net/10230/54019Data de publicació electrònica: 27-06-2022Most methods for single-cell transcriptome sequencing amplify the termini of polyadenylated transcripts, capturing only a small fraction of the total cellular transcriptome. This precludes the detection of many long non-coding, short non-coding and non-polyadenylated protein-coding transcripts and hinders alternative splicing analysis. We, therefore, developed VASA-seq to detect the total transcriptome in single cells, which is enabled by fragmenting and tailing all RNA molecules subsequent to cell lysis. The method is compatible with both plate-based formats and droplet microfluidics. We applied VASA-seq to more than 30,000 single cells in the developing mouse embryo during gastrulation and early organogenesis. Analyzing the dynamics of the total single-cell transcriptome, we discovered cell type markers, many based on non-coding RNA, and performed in vivo cell cycle analysis via detection of non-polyadenylated histone genes. RNA velocity characterization was improved, accurately retracing blood maturation trajectories. Moreover, our VASA-seq data provide a comprehensive analysis of alternative splicing during mammalian development, which highlighted substantial rearrangements during blood development and heart morphogenesis.application/pdfeng© The Author(s) 2022. 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/s41587-022-01361-8DatabasesGastrulationNon-coding RNAsRNA splicingTranscriptomicsinfo:eu-repo/semantics/openAccess