ContactPrint • DisclaimerTerms & Conditions

Recent Publications

News

  • Cappable-seq Service now available !

    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 from NEB (Ettwiller, L. et al. 2016. BMC Genomics. 17, 1 (2016), 199).

    It represents the current most sensitive and robust method for the precise identification of bacterial transcription start sites (TSSs).

vertis Biotechnologie AG
Lise-Meitner-Str. 30
D-85354 Freising
Germany

Fon +49-(0)8161-18516-11
Fax +49-(0)8161-18516-12

Contact FormHow to find us



WTSS Small RNA
dRNA-seq Cappable-seq tagRNA-seq

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).

Description

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 counterparts.

 

Example

Comparative mapping of sequence reads obtained from Cappable-seq (above panel) and from whole transcriptome cDNA libraries (lower panel).

Escherichia coli

Rhizobium leguminosarum

 

For more Information