PT  - JOURNAL ARTICLE
AU  - Veronika A. Herzog
AU  - Brian Reichholf
AU  - Tobias Neumann
AU  - Philipp Rescheneder
AU  - Pooja Bhat
AU  - Thomas R. Burkard
AU  - Wiebke Wlotzka
AU  - Arndt von Haeseler
AU  - Johannes Zuber
AU  - Stefan L. Ameres
TI  - Thiol-linked alkylation for the metabolic sequencing of RNA
AID  - 10.1101/177642
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 177642
4099  - http://biorxiv.org/content/early/2017/08/17/177642.short
4100  - http://biorxiv.org/content/early/2017/08/17/177642.full
AB  - 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 (SLAM-seq), an orthogonal chemistry-based epitranscriptomics-sequencing technology that uncovers 4-thiouridine (s4U)-incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM-seq enables rapid access to RNA polymerase II-dependent gene expression dynamics in the context of total RNA. When applied to mouse embryonic stem cells, SLAM-seq provides global and transcript-specific insights into pluripotency-associated gene expression. We validated the method by showing that the RNA-polymerase II-dependent transcriptional output scales 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. SLAM-seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective, and scalable manner.One Sentence Summary: Chemical nucleotide-analog derivatization provides global insights into transcriptional and post-transcriptional gene regulation