Degradome (t-plots). The power of coupling PARE and HTS

Degradome
sequencing is also known as parallel analysis of RNA ends (PARE) identifies miRNA
mediated mRNA cleavage products on a global scale by high-throughput sequencing
(HTS) of products from 5′ RNA ligase mediated-rapid amplification of cDNA ends
(5′ RLM-RACE) procedure (German et al. 2008). This method takes advantage of sequencing
the free 5′ -monophosphate remaining on the 3′ fragment after AGO-mediated
cleavage, to which an RNA adaptor is ligated without any additional chemical
modification. Subsequent reverse transcribed and were digested with MmeI,
purified and then 3′ adapter ligated and PCR-amplified prior to high-throughput
sequencing. Sequencing reads are mapped to mRNAs and the 5′ terminal nucleotide
of miRNA-cleaved mRNA fragments corresponds to the nucleotide that is
complementary to the 10th nucleotide of the miRNA (Fig. 1.5) (German et al. 2008). Therefore, the
cleaved RNA targets have distinct peaks in the degradome sequence reads at the
predicted cleavage site relative to other regions of the transcript, which can
be confirmed miRNA target plots (t-plots). The power of coupling PARE and HTS
has been demonstrated by identifying widespread mRNA cleavage events regulated
by miRNAs in Arabidopsis (Addo-Quaye
et al. 2008), rice (Li et al. 2010), grapevine (Pantaleo et al. 2010). Using
degradome sequencing, many of the previously validated and predicted targets of
miRNAs were verified in Arabidopsis
(Gregory et al. 2008; Addo-Quaye et al. 2008), indicating that it is an
efficient strategy to identify sRNA targets on a large scale in plants. In
addition, this technique has been successfully used for profiling of the miRNAs
target in oilseed rape (Xu et al. 2012), cucumber (Mao et al. 2012), orange
(Zhang et al. 2012), asparagus (Chen et al. 2016), apple (Song et al. 2017), miRNAs
target genes during cotton somatic embryogenesis (Yang et al. 2013), regulation
mode of miRNAs on their target mRNAs during grapevine development (Wang et al.
2014), heat responsive miRNA target in wild tomato (Zhou et al. 2016). Wang et
al. (2016) identified drought responsive miRNA target in foxtail millet.