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Thiol-linked alkylation for the metabolic sequencing of RNA [Primer Extension Assays]

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 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 provides 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.

ORGANISM(S): synthetic construct

PROVIDER: GSE99969 | GEO | 2017/09/23

SECONDARY ACCESSION(S): PRJNA390464

REPOSITORIES: GEO

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