{Reference Type}: Journal Article
{Title}: Transcriptome-Wide Profiling of RNA Stability.
{Author}: Fasching N;Petržílek J;Popitsch N;Herzog VA;Ameres SL;
{Journal}: Methods Mol Biol
{Volume}: 2404
{Issue}: 0
{Year}: 2022
暂无{DOI}: 10.1007/978-1-0716-1851-6_17
{Abstract}: 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.