RAMPAGE: Promoter Activity Profiling by Paired‐End Sequencing of 5′‐Complete cDNAs

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. Curr. Protoc. Mol. Biol . 104:25B.11.1‐25B.11.16.