GSM7090346: foxo gain-of-function, replicate 1, scRNAseq; Drosophila melanogaster; RNA-Seq
After 4 days at at 29°C, around 30 thoraces per genotype were dissected making sure the guts were completely removed, snap frozen and stored at -80°C. Single nuclei extractions were prepared according to Fly Cell Atlas protocol with few modifications (Li et al., 2022). Thoraces were homogenized in 1 ml of homogenization buffer (Li et al., 2022) using 1ml dounce (Wheaton 357538). After filtering the extract with a cell strainer (40 µm) and a 40 µm Flowmi (BelArt, H13680-0040), nuclei were collected via centrifugation for 10 minutes at 1000g at 4 ºC, and then re-suspended in 800 ul PBS/BSA buffer (1xPBS/0.5%BSA with RNase inhibitor). Nuclei solutions were filtered again using 40 µm Flowmi (BelArt, H13680-0040) and stained with DRAQ7™ Dye (Invitrogen, D15106). Single nuclei were sorted using a Sony SH800Z Cell Sorter.  Around 400k nuclei were collected, centrifuged for 1000g for 10 minutes at 4 ºC, ressuspended at 800-1000 cells/µl in PBS/BSA buffer, and then processed for snRNA-seq 10x genomics following the manufacturer instructions. Libraries were generated according to 10X genomics Next GEM Single Cell 3' Library and Gel Bead Kit v3.1 10x snRNA-seq