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

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

doi:10.1038/s41587-022-01361-8

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

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

Hubrecht Institute

Onderzoeksoutput: Bijdrage aan wetenschappelijk tijdschrift/periodieke uitgave › Artikel › Wetenschappelijk › peer review

82 Citaten (Scopus)

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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.

Originele taal-2	Engels
Tijdschrift	Nature Biotechnology
DOI's	https://doi.org/10.1038/s41587-022-01361-8
Status	E-pub ahead of print - 27 jun. 2022

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Salmen, F., De Jonghe, J., Kaminski, T. S., Alemany, A., Parada, G. E., Verity-Legg, J., Yanagida, A., Kohler, T. N., Battich, N., van den Brekel, F., Ellermann, A. L., Arias, A. M., Nichols, J., Hemberg, M., Hollfelder, F., & van Oudenaarden, A. (2022). High-throughput total RNA sequencing in single cells using VASA-seq. Nature Biotechnology. Online publicatie vooraf. https://doi.org/10.1038/s41587-022-01361-8

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title = "High-throughput total RNA sequencing in single cells using VASA-seq",

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.",

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

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year = "2022",

month = jun,

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doi = "10.1038/s41587-022-01361-8",

language = "English",

journal = "Nature Biotechnology",

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T1 - High-throughput total RNA sequencing in single cells using VASA-seq

AU - Salmen, Fredrik

AU - De Jonghe, Joachim

AU - Kaminski, Tomasz S

AU - Alemany, Anna

AU - Parada, Guillermo E

AU - Verity-Legg, Joe

AU - Yanagida, Ayaka

AU - Kohler, Timo N

AU - Battich, Nicholas

AU - van den Brekel, Floris

AU - Ellermann, Anna L

AU - Arias, Alfonso Martinez

AU - Nichols, Jennifer

AU - Hemberg, Martin

AU - Hollfelder, Florian

AU - van Oudenaarden, Alexander

PY - 2022/6/27

Y1 - 2022/6/27

N2 - 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.

AB - 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.

U2 - 10.1038/s41587-022-01361-8

DO - 10.1038/s41587-022-01361-8

M3 - Article

C2 - 35760914

SN - 1087-0156

JO - Nature Biotechnology

JF - Nature Biotechnology

ER -

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

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