Bombyx mori
Per condition, one well of a 6-well plate was seeded with 6 × 105 BmN4 cells 24h prior to transfection with either HA-eGFP, HA-BmAgo3, HA-Siwi or HA-BmGtsf1L using X-tremeGene HP transfection reagent. 72h post transfection, cells were harvested and an anti-HA immunoprecipitation was performed as described above, the experiment was performed in duplicate. Immunopurified RNAs were extracted from beads by adding 1 mL Trizol LS (Invitrogen #10296028), according to the manufacturer's instructions. The lysate was incubated at RT for 5 min to allow complete dissociation of the nucleoprotein complex. Next, 200 µL of chloroform was added to 1 mL of lysate followed by harsh mixing and centrifugation at 12,000×g for 15 min at 4°C. Another round of chloroform extraction was performed and the aqueous phase was transferred to a fresh tube to which 1 volume (500 µL) of ice-cold isopropanol was added for RNA precipitation. RNA pellets were washed twice in 1 mL of 70% ice-cold ethanol and centrifuged at 7,500×g for 10 min at 4°C. The RNA pellet was air-dried and dissolved in nuclease-free water.