Construction of Parallel Analysis of RNA Ends (PARE) libraries for the study of cleaved miRNA targets and the RNA degradome. Export

@article{citeulike:4145586, 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.}, author = {German, M. A. and Luo, S. and Schroth, G. and Meyers, B. C. and Green, P. J.}, citeulike-article-id = {4145586}, citeulike-linkout-0 = {http://dx.doi.org/10.1038/nprot.2009.8}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/19247285}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=19247285}, doi = {10.1038/nprot.2009.8}, issn = {1750-2799}, journal = {Nature protocols}, keywords = {genomics, mirna, mirna\_target, technology}, number = {3}, pages = {356--362}, posted-at = {2009-07-01 12:46:14}, priority = {2}, title = {Construction of Parallel Analysis of RNA Ends (PARE) libraries for the study of cleaved miRNA targets and the RNA degradome.}, url = {http://dx.doi.org/10.1038/nprot.2009.8}, volume = {4}, year = {2009} }

TY - JOUR ID - citeulike:4145586 L3 - citeulike-article-id:4145586 N2 - 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. IS - 3 JF - Nature protocols SN - 1750-2799 EP - 362 TI - Construction of Parallel Analysis of RNA Ends (PARE) libraries for the study of cleaved miRNA targets and the RNA degradome. VL - 4 SP - 356 KW - genomics KW - mirna KW - mirna_target KW - technology AU - German, MA AU - Luo, S AU - Schroth, G AU - Meyers, BC AU - Green, PJ PY - 2009/// UR - http://dx.doi.org/10.1038/nprot.2009.8 ER -