GSM9648491: esg-GAL4/Luciferase RNAi (control), P0 (0 hour pupa), gut; Drosophila melanogaster; RNA-Seq
Single-nucleus suspensions were prepared following the protocol we described previously [PMID: 35620068], except that homogenization was limited to 15 strokes with a tight pestle. Next, we used the BD AriaIII FACS sorter to collect nuclei. Nuclei were stained by Hoechst-33342 on ice (1:1000; >5min). Hoechst+ nuclei were collected during sorting. Individual nuclei were collected into one 1.5ml RNase-free Eppendorf tube with 200µl 1x PBS with 0.5% BSA (RNase inhibitor added) as the receiving buffer. For each sample, 100k nuclei were collected. Nuclei were spun down for 10 min at 950g at 4°C and then resuspended using 40µl or desired amount of 1x PBS with 0.5% BSA (RNase inhibitor added). 2µl of nucleus suspension was used for counting the nuclei with hemocytometers to calculate the concentration. 24K nuclei were loaded to the 10x Chromium Next GEM Chip G for each channel to target >10k nuclei. Next, we performed snRNA-seq using the 10x Genomics platform with the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index) with the following settings. All PCR reactions were performed using the BioRad C1000 Touch Thermal cycler with a 96-deep Well Reaction Module. For cDNA amplification, 12 cycles were performed. Sample index PCR was conducted following the 10x Genomics protocol recommended cycle numbers. As per the 10x protocol, 1:10 dilutions of amplified cDNA and final libraries were evaluated on a bioanalyzer. The final library was sent to Admera Health, LLC for Illumina NovaSeq X plus 25B 2x150 lane sequencing with the dual index configuration Read 1 28 cycles, Index 1 (i7) 10 cycles, Index 2 (i5) 10 cycles, and Read 2 90 cycles. A PhiX control library was spiked in at 5% concentration. The sequencing depth is about 37-56K reads per nucleus.