GSM6211400: eye_disc_tumor_late_scrna; Drosophila melanogaster; RNA-Seq
Eye-antennal discs or wing discs were dissected and transferred to a tube containing 100 µl ice cold PBS. After centrifugation at 800 g for 5 min, the supernatant was replaced by 50 µl of dispase (3 mg/ml, Sigma-Aldrich_D4818-2mg) and 75 µl collagenase I (100 mg/ml, Invitrogen_17100-017). Discs were dissociated at 25°C in a Thermoshaker (Grant Bio PCMT) for 45 min at 25°C, 500 rpm. The enzymatic reaction was reinforced by pipette mixing every 15 min. Cells were washed with 1 ml ice cold PBS and resuspended in 400 µl PBS supplemented with 0.04% BSA. Cell suspensions were passed through a 10 µM pluriStrainer (ImTec Diagnostics-435001050). Cell viability and concentration were assessed by the LUNA-FL Dual Fluorescence Cell Counter. Single-cell libraries were generated using the 10x Chromium Single-Cell Instrument and Single Cell 3' Gene Expression (GEX) kit according to the manufacturer's protocol. Briefly, single cells from eye-antennal discs or wing discs were suspended in 0.04% BSA-PBS. After generation of nanoliter-scale Gel bead-in-emulsions (GEMs), GEMs were reverse transcribed in a C1000 Touch Thermal Cycler (Bio Rad) programmed at 53°C for 45 min, 85°C for 5 min, and hold at 4°C. After reverse transcription, single-cell droplets were broken and the single-strand cDNA was isolated and cleaned with Cleanup Mix containing DynaBeads (Thermo Fisher Scientific). cDNA was then amplified by PCR: 98°C for 3 min; 12 cycles of 98°C for 15 s, 67°C for 20 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. Subsequently, the amplified cDNA was fragmented, end-repaired, A-tailed and index adaptor ligated, with SPRIselect cleanup in between steps. The final gene expression library was amplified by PCR: 98°C for 45 s; 14 cycles of 98°C for 20 s, 54°C for 30 s, 72°C for 20 s; 72°C for 1 min; and hold at 4°C. The sequencing-ready library was cleaned up with SPRIselect beads.