GSM8590671: Rrp40G146C, replicate 1, snRNAseq; Drosophila melanogaster; RNA-Seq
Two biological replicates of newly eclosed adult female flies were prepared for each biological replicate (Rrp40WT, Rrp40G11A, and Rrp40G146C). For each sample, 20 fly heads were pooled together, and nuclei were dissociated using the methods adopted from (McLaughlin et al., 2022; Teefy et al., 2022). For the nuclei dissociation, nuclei were dissociated in 1000µL of homogenization buffer (nuclease free water, sucrose, 10mM Tris (pH 8.0), 25 mM KCl, 5 mM MgCl2, 0.1% Triton-x 100, RNasin Plus, 1x protease inhibitor, and 0.1mM DTT) with a 1mL Dounce homogenizer. Homogenization was performed on ice with 20 passes of the loose Dounce and 40 passes of the tight Dounce pestle, and then filtered the solution with a 70µm cell filter into a 15mL conical precoated in 5% Bovine serum albumin. 4mL of nuclei wash buffer (0.5% BSA, PBS, and RNasin Plus) was added to the sample over the filter. Sample was centrifuged, and supernatant was discarded. Another wash step was performed with 5 mL of wash buffer and centrifuged again. Discard the supernatant, resuspend the pellet with 1mL of wash buffer, then transfer to a FACS tube. Add 600µL of debris removal solution (Miltenyi Biotec, Cat# 30-109-398), then 2mL of the resuspension buffer (0.5% BSA, PBS, RNasin) slowly on top. Samples were centrifuged and washed again as described above. Following supernatant removal, the nuclei were resuspended in 150µL of resuspension buffer using a wide-bore tip. The sample was then drawn with a standard pipette and filtered once more using a 40µm Flowmi strainer. Single-cell libraries were prepared using Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (10X Genomics, PN- 1000213) according to manufacturer's instructions (10x Genomics User Guide Chromium Next GEM Single-cell 30 Reagent Kits v3.1 (CG000204, Rev D)).