NanoCAGE: A High-Resolution Technique to Discover and Interrogate Cell Transcriptomes

Cap Analysis Gene Expression (CAGE) is a method that identifies the 5' ends of transcripts, allowing the discovery of new promoters and the quantification of gene activity. Combining together promoter location and their expression levels, the CAGE data is essential for annotation-agnostic studies of regulatory gene networks. CAGE originally requires large input amounts of RNA not obtainable from highly refined samples such as tissue microdissections and subcellular fractions. Therefore, we developed nanoCAGE to capture the 5′ ends of transcripts from as little as 10 ng of total RNA. To take advantage of the capacity of current sequencers to produce longer reads (50~100 bp), we abandoned the enzymatic cleavage step that was limiting the informative length of the reads to ~25 bp, and now prepare cap-selected cDNAs ready for direct sequencing of their 5′ end, optionally mate-paired with the 3′ end, that informs about downstream sequences. This protocol describes how to prepare nanoCAGE libraries starting from 50 ng of total RNA for sequencing on an Illumina GA II platform.