Global Run-On Sequencing (GRO-Seq).
Transcription occurring at gene loci results in accumulation of mature RNA molecules (i.e., mRNAs) that can be easily assayed by RT-PCR or RNA sequencing. However, the steady-state level of RNA does not accurately mirror transcriptional activity per se. In fact, RNA stability plays a major role in determining the relative abundance of any given RNA molecule. Here, I describe a protocol of Nuclear Run-On assay coupled to deep sequencing to assess real-time transcription from engaged RNA polymerase. Mapping nascent transcripts at the genome-wide scale provides a reliable measure of transcriptional activity in mammalian cells and delivers a high-resolution map of coding and noncoding transcripts that is especially useful for annotation and quantification of short-lived RNA molecules.
The Wistar Institute, 3601 Spruce Street, Philadelphia, PA, 19104, USA. agardini@wistar.org.
Alessandro
A
Gardini
D000818:Animals; D002874:Chromosome Mapping; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D020871:RNA Stability; D062085:RNA, Long Noncoding / Q000737:chemistry / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
D062085:RNA, Long Noncoding; D012313:RNA; D012321:DNA-Directed RNA Polymerases
Deep sequencing; Enhancer RNA; Nascent RNA; Noncoding RNA; Nuclear run-on; RNA polymerase; Transcription elongation; Transcription initiation
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