Transcriptome-Wide Profiling of RNA Stability.

Autor:	Fasching N; IMBA-Institute of Molecular Biotechnology, Vienna Biocenter (VBC), Vienna, Austria., Petržílek J; IMBA-Institute of Molecular Biotechnology, Vienna Biocenter (VBC), Vienna, Austria.; Vienna BioCenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, Vienna, Austria., Popitsch N; IMBA-Institute of Molecular Biotechnology, Vienna Biocenter (VBC), Vienna, Austria., Herzog VA; IMBA-Institute of Molecular Biotechnology, Vienna Biocenter (VBC), Vienna, Austria., Ameres SL; IMBA-Institute of Molecular Biotechnology, Vienna Biocenter (VBC), Vienna, Austria. stefan.ameres@univie.ac.at.; Max Perutz Labs, University of Vienna, Vienna Biocenter (VBC), Vienna, Austria. stefan.ameres@univie.ac.at.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2022; Vol. 2404, pp. 311-330.
DOI:	10.1007/978-1-0716-1851-6_17
Abstrakt:	Gene expression is controlled at multiple levels, including RNA transcription and turnover. But determining the relative contributions of RNA biogenesis and decay to the steady-state abundance of cellular transcripts remains challenging because conventional transcriptomics approaches do not provide the temporal resolution to derive the kinetic parameters underlying steady-state gene expression.Here, we describe a protocol that combines metabolic RNA labeling by 4-thiouridine with chemical nucleoside conversion and whole-transcriptome sequencing followed by bioinformatics analysis to determine RNA stability in cultured cells at a genomic scale. Time-resolved transcriptomics by thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAMseq) provides accurate information on transcript half-lives across annotated features in the genome, including by-products of transcription, such as introns. We provide a step-by-step instruction for time-resolved transcriptomics, which enhances traditional RNA sequencing protocols to acquire the temporal resolution required to directly measure the cellular kinetics of RNA turnover under physiological conditions. (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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