GSM7520161: LegNeu; Drosophila melanogaster; RNA-Seq
Proximal femurs from 666 flies (iav-Gal4>unc84-2xGFP; 2-3 days old) were dissected and put into 1.5ml RNAase free Eppendorf tubes, flash-frozen using liquid nitrogen, and stored at –80°C. Single-nucleus suspensions were prepared following the protocol we described previously (McLaughlin et al., 2022). Nuclei were stained by Hoechst-33342 (1:1000; >5min). Next, we collected nuclei using Fluorescence activated cell sorting (FACS). We used the BD Aria III sorter for collecting nuclei. Since the iav+ nuclei are labeled with nuclear GFP, we gated the population first by Hoechst signal and then by GFP. Nuclei were collected into a 1.5ml tube with 200ul 1x PBS with 0.5% BSA as the receiving buffer (RNase inhibitor added). In total 5,596 GFP+ nuclei were collected. Nuclei were spun down for 10 min at 1000g at 4 ̊C, and then re-suspended using 43.2ul 1x PBS with 0.5% BSA (RNase inhibitor added). We performed snRNA-seq using the 10x Genomics system with 3' v3.1 kit with following settings. All PCR reactions were performed using the Biorad C1000 Touch Thermal cycler with 96-Deep Well Reaction Module. 13 cycles were used for cDNA amplification and 16 cycles were used for sample index PCR. As per 10x protocol, 1:10 dilutions of amplified cDNA and final libraries were evaluated on Agilent TapeStation. The final library was sent to Novogene Corporation Inc. for Illumina NovaSeq PE150 S4 partial lane sequencing with the single index configuration Read 1 28 cycles, Index 1 (i7) 8 cycles, and Read 2 91 cycles.