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.