GSM9238519: glial DIP-β overexpression treatment; Drosophila melanogaster; RNA-Seq
The heads of female flies were collected at day 50 (i.e., when the flies were 50 days old), placed into 1.5 mL Eppendorf tubes, flash-frozen in liquid nitrogen, and stored at -80C. In total, two samples (each containing 20 heads) were prepared for single-nucleus RNA sequencing: one where DIP-beta had been overexpressed in glial cells from day 5-50 and one where DIP-beta expression had not been manipulated (i.e., the flies received an ethanol control instead of the Ru486 drug used to induce overexpression of DIP-beta). Single-nucleus suspensions were prepared as described in the Fly Cell Atlas (H. Li et al. 2022). After nuclei dissociation, the nuclei were purified from debris using the SONY SH800 FACS sorter. Nuclei were stained with Hoechst-33342 (1:1000, >5 minutes). It is typical to observe separate cell populations during FACS since some cells have variable sizes of nuclei and ploidy numbers. All populations were included to ensure every cell would be captured from the samples. Nuclei were collected in a 1.5 mL Eppendorf tube containing 200uL 1X PBS with 0.5% BSA (RNase inhibitor added). For each of the 10X Genomics runs, 250-300k nuclei were collected. The nuclei were centrifuged for 10 minutes at 1000g in 4C. conditions and then resuspended using 40uL of 1X PBS with 0.5% BSA (RNase inhibitor added). 2uL of the nuclei suspension were allocated for counting under a hemocytometer to calculate the nuclei concentration. For the high-throughput single-cell sequencing kit from 10X Genomics, we loaded 50k cells into the Chromium Controller to target a final number of 20-30k nuclei. Single-nucleus RNA-seq libraries were prepared using the 10X Genomics Chromium Single Cell 3' HT v3.1 protocol. All PCR reactions were performed using the BioRad C1000 Touch Thermal Cycler with the 96-Deep Well Reaction Module. We used 11 cycles for the cDNA amplification and then we used 13 cycles for the sample index PCR. As per the 10X Genomics protocol, 1:10 dilutions of amplified cDNA and the final gene expression libraries were evaluated on the Agilent Tapestation Bioanalyzer. Each library was diluted to approximately 4nM and equal volumes of each library were pooled in a NovaSeq PE150 S4 sequencing run. Pools were sequenced with the dual index configuration Read 1 28 cycles, Index 1 (i7) 10 cycles, Index 2 (i5) 10 cycles, and Read 2 90 cycles. The sequencing depth was about 30-40k reads per nucleus.