title: GSM5530529: 96h control replicate 2; Drosophila melanogaster; RNA-Seq
library_construction_protocol: For bulk-RNA sequencing: Whole ovaries were extracted from 40 females in complete medium (Grace's Insect Basal Medium supplemented with 15% FBS. The anterior part of the ovaries were carefully removed before stage 10 egg chambers. Samples were transferred to a steril Eppendorf tube and media was aspirated. Samples were flash frozen using liquid nitrogen and stored until day of the library preparation in -80°C. Total RNA was extracted using Trizol. For single-cell RNA sequencing: Whole ovaries were extracted from female flies that were dissected in complete medium (Grace's Insect Basal Medium supplemented with 15% FBS) and were transferred to a tube containing 300 μL EBSS (no calcium, magnesium, and phenol red), followed by a gentle wash for 2 minutes. The EBSS was then removed and the tissue was dissociated in 100 μL 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 μL complete medium was added to dissociated cells. The suspension was then passed through a 40 μL sterile cell strainer and centrifuged for 10 minutes at 700 RCF to remove large eggs with intact eggshell which cannot be dissociated and debris. Supernatant was removed and single cells were re-suspended in 100 μL. Cell viability was assayed using Trypan Blue and estimates of cell concentration were made using a hemocytometer. Cells were then further diluted to an approximate, final concentration of 2,000 cells/μL according to 10X Genomics recommendations. Bulk sequencing libraries were made using the Ultra II Directional RNA library prep kit for Illumina using the protocol for NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB#E7490). Single-cell libraries were prepared using the Single Cell 3' Library & Gel Bead Kit v2 and Chip Kit according to the recommended 10X Genomics protocol. Single cell suspension was loaded onto the Chromium Controller (10X Genomics). Library quantification assays and quality check analysis was performed using the 2100 Bioanalyzer instrument (Agilent Technologies). The library samples were then diluted to a 10nM concentration and loaded onto two lanes of the NovaSeq 6000 (Illumina) instrument flow cell for a 100-cycle sequencing run.