title: RNA sequencing of adult Drosophila ovary
design: Drosophila ovaries with the desired genotypes (hsFLP; FRT42D tubGAL80/FRT42DaPKCk06403; actin-GAL4 UAS-mCD8:GFP/UAS-EGFRA887T) at 6 d ACI were then dissected in complete medium (Graces Insect Basal Medium supplemented with 15% fetal bovine serum) as previously described. To prevent cell clumping, ovaries were transferred to a tube containing 300 L Earles Balanced Salt Solution (EBSS) (no calcium, magnesium, and phenol red) and gently washed for 2 minutes. The EBSS was then removed, and the tissue was dissociated in 100 L Papain (50 U/mL in EBSS and previously heat activated in 37 C for 15 minutes) for 30 minutes. The suspension was mechanically dissociated every 3 minutes by gentle pipetting up and down. To quench the digestion, 500 L complete medium was added to dissociated cells. The suspension was then passed through a 40 L sterile cell strainer and centrifuged for 10 minutes at 700 RCF to remove debris and large eggs with intact eggshells that could not be dissociated. This process also filtered out larger germline cells, which increase dramatically in size around stage 9. The supernatant was removed, and single cells were resuspended in 100L. Cell viability was assayed with AOPI solution and cell number was determined with a Nexcelom Cellometer. Cells were then further diluted to an approximate, final density of 2,000 cells/L according to 10X Genomics.