Facs arria 2 cell sorter

After injection of oncogenic cells, the flies were incubated at 25 °C for 11 d to let the injected cells grow inside the fly body. While the first isolated cell suspension was kept on ice, the remaining tissues were further homogenized in the dissociation buffer with 0.5 mg/mL liberase enzyme (Sigma) at room temperature for 15 min. The dissociated tissues were repeatedly washed by PBS, and the remaining cells were harvested. All collected cells were stained with DAPI to discriminate dead cells and applied on a BD FACS Arria II cell sorter. The sorted GFP+DAPI cells (labeled as in vivo D11 OC) and GFP-DAPI cells (labeled as Host) were immediately dissolved in TRIzol (Invitrogen) for the subsequent total RNA isolation and RNAseq analysis. Embryos from at least 500 transgenic flies (Bloomington: BL1691) were collected, bleached (3 to 5 min to remove chorion membranes) and washed with phosphate-buffered saline (PBS), then placed within a cell strainer (40 μm, Falcon) and homogenized within the PBS solution using the white end of the plunger (sometimes inside the protective cap) of an insulin injector. The embryonic cell suspension was then purified using Ficoll Paque Plus solution (GE Healthcare). After centrifugation at 400 g for 30 min, the alive cells located in the interphase were collected and resuspended in PBS.