GSM8679112: starvation day5 sample1; Drosophila melanogaster; RNA-Seq
Whole-body flies without heads were flash-frozen using liquid nitrogen and were homogenized in 1ml dounce in buffer of 250 mM sucrose, 10 mM Tris pH 8.0, 25 mM KCl, 5mM MgCl, 0.1% Triton-X, 0.5% RNasin plus (Promega, N2615), 1X protease inhibitor (Promega, G652A), 0.1 mM DTT, then filtered through 40 um cell strainer and 40 um Flowmi (BelArt, H13680-0040). Samples were centrifuged, washed, and resuspended in 1 X PBS with 0.5% BSA and 0.5% RNasin plus. The suspension was filtered again with 40 um Flowmi immediately before FACS sorting. Nuclei were stained with DRAQ7TM Dye (Invitrogen, D15106) and sorted using Sony SH800Z Cell Sorter at Systems Biology Flow Cytometry Facility at Harvard Medical School. After sorting, nuclei were collected and re-suspend at 700-800 cells/µl in 1 X PBS buffer with 0.5% BSA and 0.5% RNasin plus. snRNAseq was performed according to the 10X genomics protocol (Chromium Next GEM Single Cell 3'_v3.1_Rev_D) Libraries were generated according to 10X genomics Next GEM Single Cell 3' Library and Gel Bead Kit v3.1