GSM9789191: Sublibrary2 pool of all samples; Drosophila melanogaster; RNA-Seq
Nuclei isolation was performed using protocols adapted from McLaughlin et al., 2022. Frozen fly abdomens were quickly thawed on ice and resuspended in 950 μl of homogenization buffer (HB) consisting of: 250 mM sucrose, 10 mM Tris (pH 8.0), 25 mM KCl, 5 mM MgCl2, 0.1% Triton X-100, 1x protease inhibitor cocktail (Promega G6521), 0.1 mM DTT, 200 U/mL RNAsin Plus (Promega N2615), and 20 U/mL heat-activated DNAse I (Thermo Scientific EN0521), in nuclease free water. Resuspended abdomens were transferred to a prechilled (4°C) Wheaton dounce homogenizer (1 mL capacity; Fisher Scientific 06-434) on ice and allowed to settle at the bottom of the homogenizer. To free nuclei, the loose pestle was first used to shear the tissue with 20 half-turns while trapping the abdomens at the bottom of the dounce. This was followed by 20 strokes with the loose pestle and 40 strokes with the tight pestle. The homogenized tissue was then filtered through a SWiSH 30 micron mini cell strainer (Stellar Scientific TC70-SWM-30) and centrifuged for 10 min at 500 x g at 4°C in a fixed-angle table-top centrifuge. Following isolation, the Evercode Nuclei Fixation V3 kit (Parse Biosciences ECFN3300) was used to fix and store nuclei following manufacturer instructions. Single nuclei barcoding and library preparation with the Evercode WT Mini V3 kit (Parse Biosciences ECWT3100) was performed by the Northwestern University Center for Genetic Medicine NUseq Core facility according to manufacturer instructions.