Chemical nucleoside conversion-based measurement of RNA stability by targeted and untargeted mRNA 3´ end sequencing

Gene expression profiling by high-throughput sequencing determines changes in gene expression only at steady state but prevents our understanding of the underlying gene expression kinetics. Here, we describe a protocol that combines metabolic RNA labeling with thiol-specific chemical nucleoside conversion to determine the stability of polyadenylated RNA transcripts. And we provide a targeted mRNA 3 end library preparation protocol that enable to robustly determine the stability even of RNA transcripts that escape robust detection in untargeted libraries. The described methods enable cost-effective insights into the kinetics underlying steady-state gene expression in order to study the mechanisms underlying the regulation of gene expression at a transcript-specific and genomic scale. Mouse embryonic stem cells (mES cells) were subjected to s4U metabolic RNA labeling for 12h, followed by washout and chase using non-thiol-containing uridine. Total RNA was prepared at various time points along the chase (0h, 0.5h, 1h, 3h, 6h, and 12h). Total RNA was then subjected to alkylation and targeted RNA-seq using custom-designed primers.