Day 1: Drosophila cell transfection

Drosophila S2R+ were transfected with DNA (Yki-NTEV, 14.3.3eps-CTEV, GV-2ER, UAS-Fluc and pIZ-Rluc) using FuGENEHD transfection reagent. The cells were then bathed with the dsRNA library (Ambion) and cultured at 25 °C, 0% CO₂ for 3 days.

Day 4: Cell lysis

The culture media was aspirated and Passive Lysis Buffer was added.

Day 5: Dual-luciferase reporter assay

Firefly luciferse and Renilla luciferase activity were quantified on the Envision plate reader.