Transcript abundance can be determined by various methods, including reverse transcription (RT)-PCR, microarray analysis, sequencing of expressed sequence tags (ESTs), serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS)1, 2, most of which rely on 3’ end-related sequences. But for the identification of transcription start sites (TSSs) and their associated promoters, 5’ end-specific signature sequences are required for higher annotations of expression profiles. Therefore, we and others began cloning of short sequence tags from the 5’ ends of cDNAs, using cap analysis of gene expression (CAGE)3 and 5’-SAGE4, 5. In these techniques linkers are attached to the 5’ ends of full- length enriched cDNAs to introduce a recognition site for the restriction endonuclease Mmel adjacent to the 5’ ends. Mmel cleaves cDNAs at a sequence 20 and 18 nucleotides away (3’) from its recognition site, creating a two-base overhang. After amplification, the sequencing tags are concatenated for high- throughput sequencing (Fig. 1). Here we present a CAGE protocol that has been used extensively for high- throughput analysis of mouse and human transcripts. The method includes new features for improved library construction, such as the use of random priming6 for gene discovery from nonpolyadenylated RNA, simplified full-length cDNA enrichment by multiwell filtration-based cap-trapping and a pooling strategy for high-throughput CAGE library preparation. The application of this CAGE protocol will contribute to genome annotation, gene discovery and expression profiling7.
|Original language||English (US)|
|Number of pages||1|
|State||Published - Mar 2006|
Bibliographical noteFunding Information:
We are grateful to S. Kondo and A. Hasegawa for help with bioinformatics, and H. Sato, C. Kawazu, S. Kanagawa, M. Ohno, M. Murata, K. Nomura, Y. Tagami-Takeda and K. Hayashida for support in developing, producing and sequencing CAGE libraries. This protocol was developed with the support of the Genome Network Project, the Advanced and Innovational Research Program in Life Science and the Research Grant for RIKEN Genome Exploration Research Project, all from the Ministry of Education, Culture, Sports, Science and Technology, as well as the Strategic Programs for Research and Development of RIKEN. R.K. was supported by a fellowship from the European Union (FP5 INCO2 to Japan).
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology