Large-scale sequencing projects have revealed an unexpected complexity in the origins, structures and functions of mammalian transcripts. Many loci are known to produce overlapping coding and noncoding RNAs with capped 5′ ends that vary in size. Methods to identify the 5′ ends of transcripts will facilitate the discovery of new promoters and 5′ ends derived from secondary capping events. Such methods often require high input amounts of RNA not obtainable from highly refined samples such as tissue microdissections and subcellular fractions. Therefore, we developed nano-cap analysis of gene expression (nanoCAGE), a method that captures the 5′ ends of transcripts from as little as 10 ng of total RNA, and CAGEscan, a mate-pair adaptation of nanoCAGE that captures the transcript 5′ ends linked to a downstream region. Both of these methods allow further annotation-agnostic studies of the complex human transcriptome.
Bibliographical noteFunding Information:
This work was funded by a grant of the 6th Framework of the European Union commission to the Neuro Functional Genomics consortium, by a grant of the 7th Framework to P.C. and S.G. (Dopaminet), a Grant-in-Aids for Scientific Research (A) 20241047 for P.C. and a Research Grant for RIKEN Omics Science Center from the Japanese Ministry of Education, Culture, Sports, Science and Technology to Y.H. This project was also partially supported by the U.S. National Human Genome Research Institute grant U54 HG004557. C.P. was supported by the Japanese Society for the Promotion of Science long-term fellowship P05880. S.G. was funded by a career developmental award from The Giovanni Armenise-Harvard Foundation. We thank A. Forrest for critical discussions, M. Josserand for experimental assistance and RIKEN Genome Network Analysis Service for data production.
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology