MicroRNAs (miRNAs) are ∼21nt small RNAs that pair to their target mRNAs and in many cases trigger cleavage, particularly in plants. Although many computational tools can predict miRNA:mRNA interactions, it remains critical to validate cleavage events, due to miRNA function in translational repression or due to high rates of false positives (over 90%) for unvalidated target predictions. A few years ago, three laboratories described similar methods to validate cleavage of miRNA targets by the cloning en masse of 5' ends of cleaved or uncapped mRNAs. To take advantage of the recent progress in high-throughput sequencing technology, we have devised an updated protocol to (1) enable much faster library preparation, and (2) reduce the cost by pooling indexed samples together for sequencing. Here we provide a step-by-step protocol for PARE library construction, starting from total RNA. This protocol has been successfully used in our laboratory to validate miRNA targets in a variety of plant species. We also provide advice for troubleshooting on some common issues.
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