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.
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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