GSM8383747: Tind1 (JAMO74 in scripts); Drosophila melanogaster; RNA-Seq
Tumors were dissected on ice in Ringer buffer, rinsed twice ice-cold Ringer baths and the extracellular matrix was digested by gentle pipetting for 3-5 minutes with 20µL 5X Trypsin (PAN BIOTECH P10-022100) twice diluted. Efficient digestion was promptly checked under fluorescent dissecting microscope. Consequently, for each sample, 1X Ringer buffer was added up to 1.5mL and each tube was filtered through 30µm filters (Sysmex CellTrics). Tubes were centrifugated for 4 minutes at 1000G, at 4°C; 1mL of supernatant was discarded and the same volume of fresh 1X Ringer buffer was added. A second centrifugation (4 minutes, 1000G, 4°C) was done and after carefully removing all supernatant, pellets were gently resuspended in 30µL 1X Ringer buffer for sequencing. The whole sample preparation procedure did not exceed 20 minutes. Cell suspension was loaded into the Chromium Controller using a Chromium Next GEM Single Cell 3' v3.1 kit (10x Genomics) to generate a droplet emulsion. cDNA was purified and libraries were prepared, according to the manufacturer's recommendations. Single- cell libraries were sequenced on a NextSeq 2000 Instrument (Illumina) using a P2-100 cycles kit. FastQ files were analyzed using the Cell Ranger software (10X Genomics, version 6.0.1), including alignment, filtering and quantitation of reads on the Drosophila genome r6.42 and generation of feature-barcode matrices