293 T cells transfected with Flag-tagged plasmids or infection with viruses were co-immunoprecipitated (co-IP) by anti-FLAG affinity resin (GenScript). Briefly, 293 T cells lysates in lysis buffer (20 mM Tris–HCl [pH 7.5], 150 mM NaCl, 0.5% NP-40, 5 mM MgCl2, 10% glycerol) mixed with a protease inhibitor (Roche) were incubated with 30 μl anti-FLAG affinity resin for 4 h at 4°C in the presence or absence of 10 μg/ml RNase A (Thermo Fisher Scientific) and 5 U/ml RNase III (NEB). After five times washes with 1 × wash buffer (IBA BioTAGnology), the precipitated complexes were used to detect specific proteins by Western blotting.
For AGO-IP, cells lysates in 1 ml RIPA (Cell Signaling Technology) were precleared by incubation with 20 μl of protein A/G PLUS-Agarose (Santa Cruz Biotechnology) and 2 μg of mouse IgG (Santa Cruz Biotechnology) for 1 h at 4°C for pre-clearing. 2 μg of Anti-pan Ago antibody (Millipore) or 2 μg of mouse IgG antibodies (Santa Cruz Biotechnology) and 20 μl of protein A/G PLUS-Agarose were added into the lysates and incubated together for 4 h at 4°C followed by washing five times with 1 × wash buffer. Total RNAs were extracted from the precipitated complexes using TRIzol to construct small RNA libraries.
For AGO-IP, cells lysates in 1 ml RIPA (Cell Signaling Technology) were precleared by incubation with 20 μl of protein A/G PLUS-Agarose (Santa Cruz Biotechnology) and 2 μg of mouse IgG (Santa Cruz Biotechnology) for 1 h at 4°C for pre-clearing. 2 μg of Anti-pan Ago antibody (Millipore) or 2 μg of mouse IgG antibodies (Santa Cruz Biotechnology) and 20 μl of protein A/G PLUS-Agarose were added into the lysates and incubated together for 4 h at 4°C followed by washing five times with 1 × wash buffer. Total RNAs were extracted from the precipitated complexes using TRIzol to construct small RNA libraries.