High-throughput total RNA sequencing in single cells using VASA-seq

Salmen, Fredrik; De Jonghe, Joachim; Kaminski, Tomasz S.; Alemany, Anna; Parada, Guillermo E.; Verity-Legg, Joe; Yanagida, Ayaka; Kohler, Timo N.; Battich, Nicholas; van den Brekel, Floris; Ellermann, Anna L.; Martinez Arias, Alfonso; Nichols, Jennifer; Hemberg, Martin; Hollfelder, Florian; van Oudenaarden, Alexander

doi:http://dx.doi.org/10.1038/s41587-022-01361-8

High-throughput total RNA sequencing in single cells using VASA-seq

Citation

Salmen 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/s41587-022-01361-8

Abstract

Most 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.