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

This unit presents a protocol for RAMPAGE (RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression), a method for the genome-wide identification of transcription start sites (TSS) and the quantification of promoter activity (Batut et al., 2012). RAMPAGE is based on the synthesis of 5′-complete complementary DNAs (cDNAs) from eukaryotic total RNA samples, and their sequencing on Illumina high-throughput platforms. Previous methods for high-throughput sequencing of 5′-complete cDNAs have failed to achieve high specificity for TSS identification, and often provide only scarce sequence information in the form of 20- to 30-base “tags” (Ni et al., 2010; Valen et al., 2009). This makes their alignment to reference genomes problematic, especially when it comes to the study of repeat sequences, and yields no information regarding transcript structure. This is a major pitfall, as transcript connectivity is essential to revealing the nature of the products transcribed from individual promoters. Transcript connectivity is also key to understanding relationships between functionally related elements, such as alternative promoters. The approach we describe here (Figure 1) achieves greatly increased TSS specificity through the combination of two orthogonal enrichment strategies, namely template-switching (Hirzmann et al., 1993) and cap-trapping (Carninci et al., 1996). Template-switching makes use of unique properties of certain reverse-transcriptase enzymes to add adaptor sequences to the end of 5′-complete cDNAs, while cap-trapping is based on the biotinylation and pulldown of capped RNA molecules and their associated 5′-complete cDNAs. A streamlined protocol permits the completion of the full procedure in 2-3 days, and the addition of sequence barcodes very early in the workflow allows for very efficient multiplexing, by allowing most of the procedure to be performed on large pools of samples. The resulting libraries are suitable for paired-end sequencing on Illumina platforms (GAII, HiSeq, MiSeq), the length of sequences being only limited by the capabilities of the platform. Figure 1 RAMPAGE library preparation protocol