Abstract
We have developed a novel approach called parallel analysis of RNA ends (PARE) for high-throughput identification of microRNA (miRNA) targets and diverse applications for the study of the RNA degradome. The method described here comprises a modified 5′-rapid amplification of cDNA ends, deep sequencing of 3′ cleavage products of mRNA and bioinformatic analysis. Following RNA extraction and isolation of polyadenylated RNA, a 5′-RNA adapter that includes an MmeI recognition site is ligated to 5′-monophosphorylated products, which contain mRNA fragments generated through miRNA-induced cleavage. The ligated products are reverse-transcribed, slightly amplified and cleaved with MmeI. The 5′ equally-sized fragments are gel-selected, ligated to a 3′ double-stranded DNA adapter and PCR-amplified. Following gel purification, the products are subjected to deep sequencing. The data are then matched to cDNAs and analyzed through bioinformatics filters. We describe the high-throughput protocol in detail and indicate alternative uses for PARE. The procedure presented here can be accomplished in 6–7 d.
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Acknowledgements
We are grateful to Shawn Thatcher for help in editing the manuscript. This work was supported primarily by DOE no. DE-FG02-04ER15541 and NSF no. 0445638 (P.J.G.), with additional support from USDA no. 2007-01991 (P.J.G.), NSF no. 0548569 (P.J.G and B.C.M.), NSF no. 0321437 (B.C.M.) and NIH P20 RR16472-04.
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S.L and G.S. are employees of Illumina, which is the company that has commercialized the SBS sequencing technology. Our paper describes the use of this technology for the analysis of miRNA-target RNA pairs and the RNA degradome we demonstrated this approach in Arabidopsis, but the method has broad applicability.
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German, M., Luo, S., Schroth, G. et al. Construction of Parallel Analysis of RNA Ends (PARE) libraries for the study of cleaved miRNA targets and the RNA degradome. Nat Protoc 4, 356–362 (2009). https://doi.org/10.1038/nprot.2009.8
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DOI: https://doi.org/10.1038/nprot.2009.8
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