Mapping lncRNA-Protein Interactions

To test the interaction between the lncRNAs and either AKT or WDR26, 1 × 10⁷ CA1D were transfected with siRNAs for WDR26 or with the non-targeting siRNA as a negative control. In all, 48 h after the transfection, the cells were incubated overnight with 100 µM 4-thiouridine to allow for the UV-driven crosslinking between RNAs and interacting proteins. The cells were then lysed in 20 mM Tris–HCl at pH 7.5, 100 mM KCl, 5 mM MgCl2, and 0.5% NP-40^{50 (link),51 (link)} and the proteins of interest were immunoprecipitated using 1 µg of each of the following primary antibodies: AKT (9272 S, Cell Signaling), WDR26 (ab203345, Abcam), and normal rabbit IgG (sc-2027, Santa Cruz) as negative control. The presence of the lncRNAs in the CLIP-ed samples was assessed via qRT-PCR using the Taqman probes listed in Supplementary Table 1. To map the regions of TROLL-2 and TROLL-3 interacting with WDR26, 1 × 10⁷ CA1D were utilized. The CLIP assay was performed as above and including an RNAse T1 treatment step (1 U/µL at 22 C for 10 min)^{50 (link),51 (link)} and using 1 µg of each of the following primary antibodies: WDR26 (ab203345, Abcam) and normal rabbit IgG (sc-2027, Santa Cruz) as negative control. The presence of the lncRNA fragments in the CLIP-ed samples was assessed via qRT-PCR using the primers listed in Supplementary Table 5.