RNA-IP was performed as described previously 89. Briefly, cells stably expressing 3 × Flag-tagged AGO2 were lysed in polysome lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH 7.0), 0.5% NP-40, 1 mM DTT, 100 U/ml RNasin RNase inhibitor (Promega, N2511), 2 mM vanadyl ribonucleoside complexes solution (Sigma, 94742), 25 μl/ml protease inhibitor cocktail (Sigma, P8340)), which were then subjected to IP by using M2 agarose (Sigma, F1804) followed by washing with polysome lysis buffer four times and polysome lysis buffer plus 1 M urea four times. RNAs was released by adding 150 μl of polysome lysis buffer with 0.1% SDS and 45 μg proteinase K (Ambion, AM2548) and incubated at 50 °C for 30 min. RNA extracted with phenol-chloroform-isoamyl alcohol mixture (Sigma, 77618) was recovered by adding 2 μl GlycoBlue (15 mg/ml, Ambion, AM9516), 36 μl 3 M sodium acetate and 750 μl ethanol followed by incubation at -20 °C for overnight. Precipitated RNAs were washed with 70% ethanol, air dried, and re-suspended in RNase free water followed by DNase I (Promega, M6101) treatment to remove genomic DNA. The resultant RNAs were subjected to RT-qPCR analysis.

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