Quantification of Ago2-bound siRNA

Cells were lysed at 7 h post-transfection using ice-cold lysis buffer (50 mM Tris-HCl at pH 7.5, 200 mM NaCl, 0.5% Triton, 2 mM EDTA, 1 mg mL^-1 heparin, and protease inhibitor cocktail)^{42 (link),43 (link)}. Ago2 antibody (Wako Chemicals, 015-22031, 20 μL) was absorbed onto magnetic protein G Dynabeads (Invitrogen, 40 μL) by incubation at 4 °C for 2 h on a rotator. The beads were then washed twice with lysis buffer to remove unbound antibodies. Following washing, beads were incubated with protein lysate (360 μg) overnight at 4 °C on a rotator. Following removal of unbound proteins, bound material was eluted from beads using 50 μL of 0.1 M glycine (pH 2.3) for 15 min at RT. Eluted fractions were neutralized immediately with an equal volume of 1 M Tris-HCl (pH 8) and then treated with 20 U of proteinase K for 10 min at 65 °C^{44 (link)}. The amount of Ago2-bound siRNA was then quantified using stem loop PCR technique. For the biotin-siRNA pull down experiment, strepavidin beads were blocked and incubated with cell lysates at 4 °C for 2 h^{42 (link)}. The amount of siRNA-associated Ago2 was quantified via Western blotting.