NET-Seq

SPARE

SPARE identifies genome-wide miRNA processing intermediates in plants. SPARE infers the directionality of miRNA processing (base-to-loop or loop-to-base) by analyzing the resultant cDNA sequences. If only the first cleavage position is detected, the miRNA was processed in a base-to-loop fashion; however, if all cleavage intermediates are detected, it was processed in a loop-to-base fashion. Briefly, total RNA is depleted of rRNA, and RNA adapters are ligated to the 5' ends of uncapped RNAs. These ligated RNAs are used as the template in reverse transcription using miRNA precursor–specific primers with generic adapter tails as RT primers. The resultant cDNAs are PCR-amplified, size-selected, and sequenced.

Similar methods: PARE, 5' RACE

Pros:

• Enables genome-wide identification of miRNA intermediates
• Able to infer processing directionality through sequence analysis
• Less time-consuming than existing methods
• Optimized for plant genomes

Cons:

• Not yet adopted widely by the scientific community

• SPARE: Schapire A. L., Bologna N. G., Moro B., Zhai J., Meyers B. C. and Palatnik J. F. Construction of Specific Parallel Amplification of RNA Ends (SPARE) libraries for the systematic identification of plant microRNA processing intermediates. Methods. 2013;64:283-291

1. Ma X., Tang Z., Qin J. and Meng Y. The use of high-throughput sequencing methods for plant microRNA research. RNA Biol. 2015;12:709-719

1. Schmidt S. A., Foley P. L., Jeong D. H., et al. Identification of SMG6 cleavage sites and a preferred RNA cleavage motif by global analysis of endogenous NMD targets in human cells. Nucleic Acids Res. 2015;43:309-323