CAGE-seq
CAGE-seq (Cap analysis gene expression) captures and sequences the 5′ ends of capped RNAs by biotinylating the CAP, followed by purification with streptavidin-coated magnetic beads (1). Mapping of the resulting reads (or tags) allows for genome wide identification of transcription start sites (TSS) and their related promoter regions.
Identifying the genomic co-ordinates of CAGE tags not only detects the TSSs of known mRNA, but also mRNA from novel alternative TSSs as well as those belonging to long non-coding RNAs (lncRNA), long intergenic non-coding RNA (lincRNA) and enhancer RNAs (eRNA) (2).
The number of tags mapped at specific genomic locations also provides quantification of promoter activities. Tag quantification can provide additional support along with traditional RNA-seq in terms of gene expression analysis.
Overview: RNA is reverse transcribed into first-strand cDNA using random primers. The 7-methylguanosine RNA cap is then biotinylated. RNase digests non-hybridised, single-stranded RNAs such as incompletely reverse transcribed RNA, thus eliminating them by cap trapping. This allows the capture of complete 5’ end cDNA using streptavidin beads.The cDNA tags are processed into a library which then undergoes deep sequencing.
Applications
Accurately quantify gene by their transcription start sites to determine target proteins and action mechanisms of uncharacterized compounds.
CAGE-seq can identify novel transcripts and alternative TSSs, providing insight into gene regulation and splicing events.
Discover novel enhancer RNAs and alternative promoters for known genes to identify powerful biomarkers with clinical utility.
Measure gene promotor activity to capture subtle cellular responses induced by even low-dosage drug treatment.
Identify and quantify non-coding RNAs, such as microRNAs and long non-coding RNAs, and study their roles in gene regulation.
References
1. Carninci, P., Kvam, C., Kitamura, A., Ohsumi, T., Okazaki, Y., Itoh, M., Kamiya, M., Shibata, K., Sasaki, N., Izawa, M., Muramatsu, M., Hayashizaki, Y. and Schneider, C. (1996). High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Genomics. 37(3):327-336.
2. Thodberg, M. and Sandelin, A. (2019). A step-by-step guide to analyzing CAGE data using R/Bioconductor. F1000Res. 8:886.