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

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.

Figure 1. RAMPAGE library preparation protocol

Ribosome-depleted RNA is reverse-transcribed with random primers bearing an Illumina adaptor sequence overhang. In the conditions used, the RT enzyme will often add a few non-templated C's when it reaches the 5′ end of the template, especially if the template is capped. The template-switching oligo (TSO), which has 3 riboguanosines at its 3′, can hybridize to the terminal C's. This prompts RT to switch templates, and add the TSO sequence at the end of the newly synthesized cDNA. The TSO bears the other Illumina adaptor sequence: therefore, after RT, 5′-complete cDNAs are amplifiable, whereas non-5′-complete molecules are not. The next few steps implement the cap-trapping strategy: riboses with 2′ and 3′ free hydroxyl groups are oxidized and biotinylated, and single-stranded portions of RNA are digested by RNAseI. This leaves biotin groups only at the 5′ ends of capped transcripts hybridized to 5′-complete cDNAs, which can then be recovered on streptavidin-coated beads. After PCR amplification and size selection, the cDNAs selected by these 2 orthogonal strategies can be directly sequenced on Illumina platforms.