PARE-Seq
Parallel Analysis of RNA Ends Sequencing
Various RNA degradation processes impart characteristic sequence ends. By analyzing the cleavage sites, the degradation processes can be inferred (German et al., 2008). In PARE-Seq, the degraded uncapped mRNA is ligated to 5′ adapters containing an MmeI restriction site and reverse-transcribed. The cDNA fragments are digested with Mmel, purified, ligated to 3′ adapters, and PCR-amplified. Deep sequencing of the cDNA provides information about uncapped transcripts that undergo degradation.
Advantages:
- Maps RNA degradation
- miRNA cleavage sites are identified
- No prior knowledge of the target RNA sequence is required
Disadvantages:
- Nonlinear PCR amplification can lead to biases, affecting reproducibility
- Amplification errors caused by polymerases will be represented and sequenced incorrectly
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Reuter J. A., Spacek D. V. and Snyder M. P. High-Throughput Sequencing Technologies. Mol Cell. 2015;58:586-597
References:
Yi F., Chen J. and Yu J. Global analysis of uncapped mRNA changes under drought stress and microRNA-dependent endonucleolytic cleavages in foxtail millet. BMC Plant Biol. 2015;15:241