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Curr Protoc Mol Biol. 2013 Nov 11;104:Unit 25B.11. doi: 10.1002/0471142727.mb25b11s104.

RAMPAGE: promoter activity profiling by paired-end sequencing of 5'-complete cDNAs.

Author information

1
Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

Abstract

RNA annotation and mapping of promoters for analysis of gene expression (RAMPAGE) is a method that harnesses highly specific sequencing of 5'-complete complementary DNAs to identify transcription start sites (TSSs) genome-wide. Although TSS mapping has historically relied on detection of 5'-complete cDNAs, current genome-wide approaches typically have limited specificity and provide only scarce information regarding transcript structure. RAMPAGE allows for highly stringent selection of 5'-complete molecules, thus allowing base-resolution TSS identification with a high signal-to-noise ratio. Paired-end sequencing of medium-length cDNAs yields transcript structure information that is essential to interpreting the relationship of TSSs to annotated genes and transcripts. As opposed to standard RNA-seq, RAMPAGE explicitly yields accurate and highly reproducible expression level estimates for individual promoters. Moreover, this approach offers a streamlined 2- to 3-day protocol that is optimized for extensive sample multiplexing, and is therefore adapted for large-scale projects. This method has been applied successfully to human and Drosophila samples, and in principle should be applicable to any eukaryotic system.

KEYWORDS:

RAMPAGE; expression profiling; high-throughput sequencing; promoter; transcription start site

PMID:
24510412
PMCID:
PMC4372803
DOI:
10.1002/0471142727.mb25b11s104
[Indexed for MEDLINE]
Free PMC Article
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