Double-stranded RNA (dsRNA) was synthesised using the MEGAscript T7 kit (AMB1334, Invitrogen) according to manufacturer’s instructions. In brief, dsRNA was generated using T7 Polymerase in vitro transcription from PCR amplicons obtained with T7 minimal promotor containing primers using a cDNA template from S2[Cas9] cells. 10-15 μg of dsRNA was added to 0.3x106 S2[Cas9] cells in a total of 3 ml Schneider’s Drosophila Medium. For different cell numbers, the amount of dsRNA and medium was scaled accordingly. Cells were harvested for RNA isolation four days post transfection and for cell cycle analysis and protein extraction three days post transfection.

To monitor proliferation, cells were re-seeded immediately after transfection. The cell density was determined from three independent dsRNA transfections every 24 hours using a hemocytometer. Cell viability was determined four days post transfection by measuring cell dilutions on a CASY Cell Analyser (OMNI Life Science).

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