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Nat Methods. 2010 Dec;7(12):995-1001. doi: 10.1038/nmeth.1529. Epub 2010 Nov 7.

FragSeq: transcriptome-wide RNA structure probing using high-throughput sequencing.

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  • 1Howard Hughes Medical Institute, University of California Santa Cruz, Santa Cruz, California, USA.

Abstract

Classical approaches to determine structures of noncoding RNA (ncRNA) probed only one RNA at a time with enzymes and chemicals, using gel electrophoresis to identify reactive positions. To accelerate RNA structure inference, we developed fragmentation sequencing (FragSeq), a high-throughput RNA structure probing method that uses high-throughput RNA sequencing of fragments generated by digestion with nuclease P1, which specifically cleaves single-stranded nucleic acids. In experiments probing the entire mouse nuclear transcriptome, we accurately and simultaneously mapped single-stranded RNA regions in multiple ncRNAs with known structure. We probed in two cell types to verify reproducibility. We also identified and experimentally validated structured regions in ncRNAs with, to our knowledge, no previously reported probing data.

PMID:
21057495
[PubMed - indexed for MEDLINE]
PMCID:
PMC3247016
Free PMC Article

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