Generation of cDNA Libraries from RNP-Derived Regulatory Noncoding RNAs

Next-generation sequencing of noncoding RNA (ncRNA) libraries has become an essential tool for the profiling of ncRNAs and the identification of novel ncRNA species. Here, we describe the generation of a ncRNA-derived complementary DNA (cDNA) library by 3′-tailing of ncRNAs by CTP and poly(A) polymerase, followed by 5′-adapter ligation by T4 RNA ligase and reverse transcription of ncRNAs with an oligo-d(G) anchor primer. Preliminary selection of ncRNAs from ribonucleoprotein particles (RNPs) enables a strong enrichment of the generated libraries with functional regulatory ncRNAs compared to classical approaches.