Immunopurification and RNA Analysis of PIWI Proteins

100 ovaries per sample were used in Aub and Piwi IP experiments and processed, as previously described (46 (link)). For Ago3 IP experiments, we used 150 ovaries per sample with a slight modification in the protocol. Ovary lysates were first incubated with 4 μg Ago3-380 antibody for 2 h at 4°C and then mixed with buffer-equilibrated Protein G Dynabeads (Thermo Fisher Scientific) for 90 min at 4°C. The RNAs associated with immunopurified PIWI proteins were extracted with TRIzol reagent (Ambion) and dephosphorylated with Quick CIP (NEB) in CutSmart buffer for 10 min at 37°C. After enzyme inactivation for 2 min at 80°C, a T4 PNK (NEB) labeling reaction was set in 1× CutSmart buffer with the addition of DTT to 5 mM in the presence of ^γ32PATP. Reactions were run with 8 M urea 15% PAGE.