title: GSM9301467: pannier-Gal4 domain warts knock-down; Drosophila melanogaster; RNA-Seq
library_construction_protocol: Single-nucleus suspensions were prepared following the protocol described previously [PMID: 35620068] with the adaptation of 20 strokes with the loose Dounce pestle and 20 strokes with the tight Dounce pestle120. 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 as the receiving buffer (RNase inhibitor added). For each 10x Genomics run, 100k nuclei were collected. Nuclei were spun down for 10 min at 950g at 4°C and then resuspended using 80µl 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. We loaded 40K nuclei to the 10x controller to target >20k nuclei for each channel. Next, we performed snRNA-seq using the 10x Genomics platform with the Chromium Next GEM Single Cell 3' HT (high-throughput) 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. The recommended cycle numbers from the 10x protocol were used for cDNA amplification and sample index PCR. 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 Novogene Corporation Inc. for Illumina NovaSeq PE150 S4 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 0.2 to 1% concentration. The sequencing depth is about 26-30K reads per nucleus.