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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

cDNA Synthesis From bacterial small RNA

Regulatory RNAs in prokaryotes are referred to as small RNA (sRNAs). The sRNAs are typically between 50 and 300 nt in length. We offer well established methods for the deep sequencing of bacterial small RNomes. 

References

Köhler, K. et al. 2015. Structural and mechanistic characterization of 6S RNA from the hyperthermophilic bacterium Aquifex aeolicus. Biochimie. 117, (2015), 72–86.

Madhugiri, R. et al. 2012. Small RNAs of the Bradyrhizobium/Rhodopseudomonaslineage and their analysis. RNA Biology. 9, 1 (2012), 47–58.

Wilms, I. et al. 2012. Deep sequencing uncovers numerous small RNAs on all four replicons of the plant pathogen Agrobacterium tumefaciens. RNA Biology. 9, 4 (2012), 446–57.

Chao, Y. et al. 2012. An atlas of Hfq-bound transcripts reveals 3' UTRs as a genomic reservoir of regulatory small RNAs. The EMBO Journal. 31, 20 (2012), 4005–4019.

Bohn, C. et al. 2010. Experimental discovery of small RNAs in Staphylococcus aureus reveals a riboregulator of central metabolism. Nucleic Acids Research. 38, 19 (2010), 6620–6636.

Example: Preparation of cDNA from Dickeya dadantii small RNA fraction

The small RNA fraction (< 200 nt) was isolated from Dickeya dadantii total RNA (Fig. 1). From the small RNA fraction, rRNA molecules were depleted.

 

For enrichment of sRNA molecules carrying 5' triphosphate, one half of the sRNA preparation was further treated with Terminator exonuclease (TEX) and the other half was left untreated. Then, from both samples the 5'PPP structures were coverted to 5'P using tobacco acid pyrophosphatase (TAP).

Oligonucleotide adapters were ligated to the 5' and 3' ends of the RNA samples. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3‘ adapter as primer. The resulting cDNA was amplified with PCR using a high fidelity DNA polymerase (Fig. B).

The cDNA libraries were paired-end sequenced (2x75 bp) on a Illumina NextSeq 500 system.

 
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