Differential RNA-sequencing (dRNA-seq)
Differential RNA-seq for the annotation of transcriptional start sites according to Sharma CM et al. 2010. Nature
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.
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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