In this webinar you will have a chance to learn about a new metabolic RNA labeling method SLAMseq (Herzog et al., Nature Methods, DOI: 10.1038/nmeth.4435) from its inventor.
Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAMseq), an orthogonal-chemistry-based RNA sequencing technology that detects 4-thiouridine (s4U) incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAMseq enabled rapid access to RNA-polymerase-II-dependent gene expression dynamics in the context of total RNA. We validated the method in mouse embryonic stem cells by showing that the RNA-polymerase-II-dependent transcriptional output scaled with Oct4/Sox2/Nanog-defined enhancer activity, and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAMseq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective and scalable manner.