Transcriptome-Wide Profiling of RNA Stability.
Methods in molecular biology (Clifton, N.J.)(2022)
摘要
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.
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关键词
4-Thiouridine,Gene regulation,Metabolic RNA sequencing,RNA stability,SLAMseq
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