Cappable-seq or cappable-sequencing is a technique that allows the capture of the 5′-end of primary transcripts. This method developed by Ettwiller et al. in 2016 enables the robust determination of transcription start sites (TSS) genome-wide at single-base resolution. Furthermore, cappable-seq depletes ribosomal RNA, thereby reducing the complexity of the transcriptome to a single quantifiable tag per transcript for digital profiling of gene expression in any microbiome. Cappable-seq allows isolation of primary transcripts via enzymatically capping of 5′-triphosphorylated RNA with a biotinylated GTP using the vaccinia capping enzyme. In addition, 3′-OH modifications of the GTP ribose are also acceptable substrates for the enzyme.
Cappable-seq reactions result in the specific labeling of 5′-di-, or triphosphorylated RNA ends, whereas 5′-monophosphorylated RNA ends characteristic of processed transcripts are not labeled. Furthermore, biotinylated RNA can be captured on streptavidin beads and isolated.
Reference
Ettwiller L, Buswell J, Yigit E, Schildkraut I. A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome. BMC Genomics. 2016 Mar 8;17:199. doi: 10.1186/s12864-016-2539-z. PubMed PMID: 26951544; [PubMed Central PMCID: PMC4782308].
---...---