AGO2-RIP of miRNA Targets in PANC-1 Cells

PANC-1 cells were seeded in 15 cm dishes (5 × 10⁶ cells/dish; 3 dishes were used for each antibody tested) and transfected with 0.5 nM of pre-NC, pre-miR-100 and pre-miR-125b for 24 h. AGO2-RIP was carried out as previously described by our group^{48 (link)}. Briefly, following transfection cells were washed in cold PBS, scraped in PBS and collected by centrifugation. Pellets were then resuspended in lysis buffer (20 mM Tris-HCl pH7.5, 150 mM KCl, 0.5% NP40, 2 mM EDTA, 1 mM NaF, 0.5 mM DTT, 160 U ml⁻¹ RNAsin and protease and phosphates inhibitors) and pre-cleared with Protein G sepharose beads (Sigma) for 2 h at 4°C. Part of cleared lysates (10%) was used as input and the remainder were incubated with Protein G sepharose beads conjugated with anti-AGO2 (11A9, SAB4200085, Sigma-Aldrich) or anti-IgG (Sigma-Aldrich) for 4 h at 4°C. After washing, 10 μl of the immunoprecipitate was kept for western blot analysis and the remainder was treated with DNAse I and proteinase K for 20 min at room temperature. RNA was extracted using phenol/ chloroform and and ethanol/sodium acetate precipitation. RNA was then quantified using Nanodrop.