RNA from U4.4 cells was extracted (TRIzol, Thermo Fisher) and cDNA generated using the Maxima First Strand cDNA Synthesis Kit with random primers (Thermo Fisher). A 686 bp Ago-2 region was amplified from the cDNA using specific T7-promoter flanked primers (T7-Ago2-F and T7-Ago2-R, Supplementary Table 3). PCR products were cleaned up (NucleoSpin Gel and PCR Clean-up, Macherey Nagel) and used for dsRNA production (MEGAScript RNAi kit, Thermo Fisher). Knock-down of Ago-2 was performed by transfection of 250 ng dsRNA per well in sub-confluent U4.4 cells seeded in a 48 well plate (Lipofectamine LTX, Thermo Fisher). Control knock-downs were carried out using the control dsRNA provided in the MEGAScript RNAi kit.

The efficiency of Ago-2 knockdown was assessed by mRNA quantification. Briefly, 24 h following dsRNA transfection, cells were lysed in RNA lysis buffer and RNA extracted using the Quick-RNA 96 kit (Zymo), including the in-column DNAse digestion step to remove genomic DNA. RT-qPCR was carried out using the Luna Universal One-Step RT-qPCR kit (New England Biolabs) following the manufacturer’s instructions, with Ago-2 and actin specific primers (Supplementary Table 3), on a Step-One-Plus Real-Time PCR thermocycler (Applied Biosystems). Quantification of Ago-2 mRNA expression was calculated relative to actin mRNA using the delta-delta Ct method.

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