Location: Plant Gene Expression CenterTitle: Preparation of cDNA Library for dRNA-seq Author
Submitted to: Bio-protocol
Publication Type: Other
Publication Acceptance Date: 12/1/2012
Publication Date: 12/5/2012
Publication URL: http://www.bio-protocol.org/wenzhang.aspx?id=302
Citation: Li, F., Baker, B.J. 2012. Preparation of cDNA Library for dRNA-seq. Bio-protocol. Vol 70, Pages 891-901. Interpretive Summary: MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) inhibit gene expression in eukaryotic organisms by guiding protein complexes to complementary mRNA for cleavage of miRNA target transcripts. In plants, bioinformatic analyses is used to predict miRNA target transcripts. Bioinformatic predictions of verify bioinformatic predictions of miRNA silencing of specific mRNAs and identify new targets of known miRNAs. Here we describe a protocol for preparation of plant complementary DNA (cDNA) libraries of miRNA cleaved transcripts, termed degradome RNA (dRNA) for high-through-put sequencing (dRNA-seq) for confirmation of miRNA activity. Using this protocol we generated and sequenced cDNA libraries corresponding to dRNAs of three species of Solanaceae, including tobacco, tomato and potato. We verified these sequenced dRNA sequence datasets by identifying known mRNA cleavage products of conserved miRNAs.
Technical Abstract: 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.