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microRNAs (miRNAs) are ubiquitous regulators of gene expression in eukaryotic organisms, which guide Argonaute proteins (AGO) to cleave target mRNA or inhibit its translation based on sequence complementarity. In plants, miRNA directed cleavage occurs on the target mRNA at about 10 to 11 nucleotide (nt) up stream to the site where the 5’ end of miRNA binds. Sequencing of the miRNA directed cleavage products (degradome) is widely employed as a way to both verify bioinformatic predictions of miRNA mediated regulation and identify novel targets of known miRNAs. Here we describe a protocol for preparation of degradome RNA complementary DNA library for high-through-put sequencing (dRNA-seq) using Illumina GA II sequencing platform, which is currently most popular and cost-effective. Using this protocol we successfully generated three dRNA-seq libraries using three solanaceae plants, including tobacco, tomato and potato. Although this protocol was developed with single-plexed adapter, it should be able to generate multiplexed libraries by replacing the 3’ adapter with multiplexing compatible 3’ adapter and replacing the PCR primer with indexed primers.
Materials and Reagents
The principle and application of this protocol were briefly described in Li et al. (2012). This work was supported by the National Science Foundation Plant Genome Research Program Grant (DBI-0218166) and the United States Department of Agriculture (CRIS 5335-22000-007-00D). The authors declare no conflict of interest.
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