Abstract
Cap analysis gene expression (CAGE) is a method to identify the 5' ends of transcripts, allowing the discovery of new promoters and the quantification of gene activity. Combining promoter location and their expression levels, CAGE data are essential for annotation-agnostic studies of regulatory gene networks. However, CAGE requires large amounts of input RNA, which usually are not obtainable from highly refined samples such as tissue microdissections or subcellular fractions. The nanoCAGE method can capture the 5' ends of transcripts from as little as 10 ng of total RNA and takes advantage of the capacity of current sequencers to produce longer (50-100 bp) reads. The method prepares cap-selected cDNAs ready for direct sequencing of their 5' ends (optionally mate-paired with the 3' end) that can provide information about downstream sequences. This protocol describes how to prepare nanoCAGE libraries from as little as 50 ng of total RNA within two working days. The libraries can be sequenced using an Illumina sequencer Genome Analyzer IIX [corrected] with a level of sensitivity 1000 times higher than CAGE.