GSM8994751: Pnvp Control EC enriched P8; Mus musculus; RNA-Seq
The cell suspension was washed with a FACS buffer (PBS containing 1% FBS and 0.2% EDTA). Cells were stained for 30 minutes at room temperature with PE anti-mouse CD140b Antibody (Biolegend, 136006); APC-conjugated anti-mouse CD31 antibody (BD Biosciences, 551262) and FITC-conjugated anti-mouse CD45 antibody (BioLegend, 103108) in Fc block solution (5 µg/mL CD16/CD32 Fc Block, eBioscience, 16-0161-82). After washing, cells were stained with Live/Dead Fixable Aqua Dead Cell Stain (ThermoFisher Scientific, L34957) for 20 minutes at room temperature. Stained cells were washed and resuspended in PBS. The gating strategy was defined using unstained and single-stained controls. Dead cells and doublets were excluded, and endothelial cells (CD45⁻ CD31⁺) were sorted using a BD FACS Aria Fusion. Cells were resuspended in PBS containing 0.04% BSA. Cells from WT PNVP/brain and mutant PNVP/brain sample were then subjected to single-cell RNAseq aiming at a yield of 5 000 cells per condition. Four libraries (CTL PNVP, CTL BRAIN, mutant PNVP, mutant BRAIN) were generated using the Chromium  Single Cell 3' Reagent Kits v3.1 (10x Genomics; Pleasanton, CA, USA).