RNA extraction and qRT-PCR of Drosophila S2 cells

Total RNA was extracted using the EZNA Total RNA Kit I (Omega Bio-tek) following the manufacturer’s protocol. cDNA was generated with random hexamer primers and the RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific) using 1 μg of the total RNA as template, following the manufacturer’s protocol. cDNA was diluted five times and used as templates for qRT-PCR on a LightCycler 480 Instrument II (Roche) using LightCycler 480 SYBR Green I Master reaction mix (Roche) according to the manufacturer’s instructions and the following qPCR program: (i) 95°C for 5 min, (ii) 95°C for 10 s, (iii) 55°C for 10 s, (iv) 72°C for 10 s, (v) plate read, and (vi) repeat step 2 44X. Primers used for qRT-PCR are listed in table S1. Normalization was performed relative to the housekeeping gene RpL32.