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Nat Protoc. 2011 Feb;6(2):166-74. doi: 10.1038/nprot.2010.186. Epub 2011 Jan 27.

cDNA library generation from ribonucleoprotein particles.

Author information

1
Innsbruck Medical University, Innsbruck Biocenter, Division for Genomics and RNomics, Innsbruck, Austria. mathieu.rederstorff@maem.uhp-nancy.fr

Abstract

Most, if not all, known noncoding RNAs (ncRNAs) are associated with RNA binding proteins, thus forming ribonucleoprotein particles or RNPs. Here we describe a protocol for the generation of a specialized cDNA library from RNPs, thereby increasing the proportion of functional ncRNA species in the library. To that end, cellular extracts are fractionated on 10-30% glycerol gradients. Subsequently, RNP-derived ncRNAs are isolated and 3'-tailed by cytidine triphosphate and poly(A) polymerase; this is followed by 5' adapter ligation by T4 RNA ligase. Reverse transcription of ncRNAs into cDNAs is carried out with an oligo-d(G) anchor primer. The generated cDNA libraries are subsequently submitted to high-throughput sequencing. This RNP selection procedure increases the probability of the presence of biologically relevant ncRNA species in the library compared with libraries generation methods that use size-selected, protein-devoid ncRNAs. The protocol enables the generation of deep-sequencing-compatible cDNA libraries that code for functional ncRNAs within 1 week.

PMID:
21293458
DOI:
10.1038/nprot.2010.186
[Indexed for MEDLINE]

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