Detection of RNA–DNA binding sites in long noncoding RNAs

Triple helix is a potential mechanism how lncRNAs interact with the genome. Our in silico method (Triplex Domain Finder) is able to predict the binding sites of lncRNA MEG3 and GATA6-AS in the genome. In order to validate these predictions, we develop DBD-Capture-Seq to capture the DNA loci where the given RNA oligo binds to via triplex. Method: Different RNA oligos are used The biotinylated RNA oligos (MEG3 TFR1, MEG3 TFR2, and GATA6-AS TFR1) were incubated with sheared genomic DNA to allow for triplex formation. After binding to streptavidin-coated beads, RNA-associated DNA was eluted and subjected to deep sequencing. Control experiments were conducted in the absence of biotinylated RNA oligos. Overall design: We have used 3 RNA oligos (MEG3 TFR1, MEG3 TFR2 and GATA6-AS TFR1) for capturing their triplex-forming loci (all in duplicates). Input and control experiments were tested as well.