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  • 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).

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

Differential RNA-sequencing (dRNA-seq)

Differential RNA-seq for the annotation of transcriptional start sites according to Sharma CM et al. 2010. Nature 464:250-255.

Description

Discrimination of primary from processed 5' ends by sequencing differential cDNA library pairs: one library from untreated bacterial RNA, and the other enriched for primary transcripts by terminator exonuclease treatment that degrades 5'PPP to 5'P.

The following steps are carried out during dRNA-seq cDNA library preparation:

  • Removal of rRNA molecules with rRNA depletion kit (e.g. Ribo-Zero bacteria). Also total RNA can be used for dRNA-seq, but the results are cleaner when using ribodepleted RNA.
  • Removal of processed transcripts with 5'P ends: treatment of half of the RNA sample with 5‘-phosphate-dependent Terminator RNA exonuclease (TEX; Epicentre), the other half is kept untreated.
  • Enzymatic conversion of 5' triphosphate (5'PPP) to 5' monophosphate (5'P) of both samples.
  • Ligation of RNA adapter to 5' monphosphorylated RNA.
  • cDNA library preparation according to our small RNA protocol.

References

Papenfort, K. et al. 2015. Differential RNA-seq of Vibrio cholerae identifies the VqmR small RNA as a regulator of biofilm formation. Proceedings of the National Academy of Sciences. 112, 7 (2015), E766–E775.


Bischler, T. et al. 2015. Differential RNA-seq (dRNA-seq) for annotation of transcriptional start sites and small RNAs in Helicobacter pylori. Methods. 86, (2015), 89–101.


Kopf, M. et al. 2015. Variations in the non-coding transcriptome as a driver of inter-strain divergence and physiological adaptation in bacteria. Scientific Reports. 5, (2015), 9560.


Creecy, James P., and Tyrrell Conway. "Quantitative bacterial transcriptomics with RNA-seq." Current opinion in microbiology 23 (2015): 133-140.


Thomason, M. et al. 2014. Global transcriptional start site mapping using dRNA-seq reveals novel antisense RNAs in Escherichia coli. Journal of Bacteriology. 197, 1 (2014), JB.02096–14.


Kopf, M. et al. 2014. Comparative Analysis of the Primary Transcriptome of Synechocystis sp. PCC 6803. DNA Research. 21, 5 (2014), dsu018.


Kröger, Carsten, et al. "An infection-relevant transcriptomic compendium for Salmonella enterica Serovar Typhimurium." Cell host & microbe 14.6 (2013): 683-695.


Mitschke, Jan, et al. "Dynamics of transcriptional start site selection during nitrogen stress-induced cell differentiation in Anabaena sp. PCC7120." Proceedings of the National Academy of Sciences 108.50 (2011): 20130-20135.


Sharma, Cynthia M., et al. "The primary transcriptome of the major human pathogen Helicobacter pylori." Nature 464.7286 (2010): 250-255.


Advantage of method

Well established and proven method which has been continuously validated over the last 5 years.

 
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