Construction of parallel analysis of rna ends (Pare) libraries for the study of cleaved mirna targets and the rna degradome

Marcelo A. German; Shujun Luo; Gary Schroth; Blake C. Meyers; Pamela J. Green

doi:10.1038/nprot.2009.8

Construction of parallel analysis of rna ends (Pare) libraries for the study of cleaved mirna targets and the rna degradome

Marcelo A. German, Shujun Luo, Gary Schroth, Blake C. Meyers, Pamela J. Green

Research output: Contribution to journal › Article › peer-review

271 Scopus citations

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' doublestranded 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.

Original language	English
Pages (from-to)	356-362
Number of pages	7
Journal	Nature Protocols
Volume	4
Issue number	3
DOIs	https://doi.org/10.1038/nprot.2009.8
State	Published - Mar 2009

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@article{0ea3191008664a83a70d8c10b88279bb,

title = "Construction of parallel analysis of rna ends (Pare) libraries for the study of cleaved mirna targets and the rna degradome",

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{\textquoteright}-rapid amplification of cDNA ends, deep sequencing of 3{\textquoteright} cleavage products of mRNA and bioinformatic analysis. Following RNA extraction and isolation of polyadenylated RNA, a 5{\textquoteright}-RNA adapter that includes an MmeI recognition site is ligated to 5{\textquoteright}- 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{\textquoteright} equally-sized fragments are gel-selected, ligated to a 3' doublestranded 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, \{Marcelo A.\} and Shujun Luo and Gary Schroth and Meyers, \{Blake C.\} and Green, \{Pamela J.\}",

note = "Funding Information: ACKNOWLEDGMENTS 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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AU - German, Marcelo A.

AU - Luo, Shujun

AU - Schroth, Gary

AU - Meyers, Blake C.

AU - Green, Pamela J.

N1 - Funding Information: ACKNOWLEDGMENTS 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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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' doublestranded 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.

AB - 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' doublestranded 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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DO - 10.1038/nprot.2009.8

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Construction of parallel analysis of rna ends (Pare) libraries for the study of cleaved mirna targets and the rna degradome

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