title: cDNA library generation from ribonucleoprotein particles.
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.
authors.affiliation: Innsbruck Medical University, Innsbruck Biocenter, Division for Genomics and RNomics, Innsbruck, Austria. mathieu.rederstorff@maem.uhp-nancy.fr
authors.forename: Mathieu
authors.initials: M
authors.lastname: Rederstorff
authors.forename: Alexander
authors.initials: A
authors.lastname: Hüttenhofer
mesh_terms: D002499:Centrifugation, Density Gradient; D005591:Chemical Fractionation; D018076:DNA, Complementary / Q000737:chemistry; D015723:Gene Library; D023281:Genomics / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D016133:Polymerase Chain Reaction; D048348:Reverse Transcription; D012261:Ribonucleoproteins / Q000737:chemistry*
chemical_list: D018076:DNA, Complementary; D012261:Ribonucleoproteins