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Nat Methods. 2017 Dec;14(12):1198-1204. doi: 10.1038/nmeth.4435. Epub 2017 Sep 25.

Thiol-linked alkylation of RNA to assess expression dynamics.

Author information

1
Institute of Molecular Biotechnology, Vienna Biocenter Campus, Vienna, Austria.
2
Research Institute of Molecular Pathology, Vienna Biocenter Campus, Vienna, Austria.
3
Center for Integrative Bioinformatics Vienna, Max F Perutz Laboratories, Medical University of Vienna, University of Vienna, Vienna Biocenter Campus, Vienna, Austria.

Abstract

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 RNA sequencing technology that detects 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 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. SLAM seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective and scalable manner.

PMID:
28945705
PMCID:
PMC5712218
DOI:
10.1038/nmeth.4435
[Indexed for MEDLINE]
Free PMC Article

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