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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 FL-cDNA Normalization MicroRNA PARE DGE

Parallel Analysis of RNA Ends (PARE)

For high-throughput identification of microRNA (miRNA) targets (degradome analysis), as described by German et al. 2009, NATURE PROTOCOLS, VOL.4 NO.3.

We have adapted the method to the new Illumina sequencing systems and it works very efficiently and reliable in our hands. The quality of the first reads on Illumina systems is restricted, therefore we use paired-ends (PE) sequencing in order to unequivocally determine the sequence of the miRNA procession sites.

 

References

Surbanovski, Nada, et al. "A highly specific microRNA-mediated mechanism silences LTR retrotransposons of strawberry." The Plant Journal 85.1 (2016): 70-82.

  

Example: Preparation of PARE cDNA from Nicotiana

From 10 µg of the total RNA from Nicotiana spec. (Fig. 1, panel A), poly(A)+ RNA was isolated which was used for cDNA synthesis.
 
 
 
First, an RNA adapter (modified Solexa Gex 1 adapter) was ligated to the 5'-phosphate of processed mRNA. First-strand cDNA synthesis was primed with an oligo-dT adapter primer and the cDNA was amplified with PCR in 12 cycles (Fig.1, panel B).

The cDNA was further treated with Mme I, which cuts 20 bp downstream of its recognition site TCCRAC, located at the 3‘ end of the ligated Solexa Gex 1 adapter. To the released 51 bp long Mme I fragments, the Illumina 3' TruSeq adapter was ligated. The ligation products were purified and PCR amplified in 5 cycles (Fig 1, panel C). 

The PARE cDNA was paired-end sequenced on an Illumina NextSeq 500 System using 2x25 bp read length.
 
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