Cappable-seq for the precise identification of transcription start sites (TSSs)
In close cooperation with New England Biolabs, Inc. (NEB), VERTIS is now able to make the new Cappable-seq
method available to its customers. The method was developed by Laurence
Ettwiller and Ira Schildkraut (Ettwiller, L. et al. 2016. BMC Genomics.
17, 1 (2016), 199).
Cappable-seq is currently the most sensitive and robust method for the precise identification of bacterial transcription start sites (TSSs).
The bacterial primary transcripts carry a distinctive 5' triphosphorylated end that distinguishes these transcripts from all other RNA species. The currently available methods do not use targeted enrichment for the 5' end of primary transcripts, but rather attempt to deplete non-targeted RNA, like 5'-phosphate-dependent exonuclease (TEX) treatment of RNA (dRNA-seq).
Cappable-seq directly targets the 5' end of primary transcripts, enabling determination of transcription start sites at single base resolution. This is achieved by labelling the 5' triphosphorylated end of RNA with a biotin derivate.
The following steps are carried out during Cappable-seq cDNA library preparation:
- Capping of the 5' triphosphorylated RNA with 3'- desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) (NEB) using the vaccinia capping enzyme (VCE) (NEB) for reversible binding of biotinylatid RNA species to streptavidin
- Capturing of biotinylated RNA species on streptavidin beads and elution to obtain the 5' fragment of the primary transcripts
- Enzymatic conversion of 5' triphosphate (5'PPP) to 5' monophosphate (5'P)
- Ligation of an RNA adapter to the formed 5'P of primary transcripts
- cDNA library preparation using our random or small RNA protocol
Advantage of method
The method directly targets 5'PPP in total RNA preparations and there is no need the perform rRNA depletion beforehand. Therefore, there are no restrictions towards organisms, for which no feasible RNA depletion kit is available.
As a consequence, Cappable-seq has a broad range of applications, offering the ability to
investigate the triphosphorylated population of RNA molecules that would
otherwise be masked by the overwhelming majority of their processed
Comparative mapping of sequence reads obtained from Cappable-seq (above panel) and from whole transcriptome cDNA libraries (lower panel).
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