Purification and Retroviral Transduction of LSK Cells

LSK cells were purified as described previously (61 (link)). In brief, BM cells were isolated from femora and tibiae of mice in PBS containing 0.5% BSA and 2 mM EDTA buffer. Lineage negative cells (Lin-) were isolated using the Lineage Cell Depletion Kit (Miltenyi Biotec). Lin- cells were stained with APC-c-Kit and PE-Sca1 antibodies, and LSK cells were purified with a BD FACSAria Fusion cytometer and cultured in SFEM medium plus 10% FBS supplemented with 20 ng/mL Flt3L, 20 ng/mL IL-6, 100 ng/mL SCF, 20 ng/mL TPO, and 0.1 mM β-ME. After 48 hours of culture, LSK cells were spin-infected with retroviruses expressing WT or Q61R mutant NRAS preloaded on RetroNectin- coated plates, with 10 μg/ml polybrene (Sigma-Aldrich). Cells were either transplanted 24 hours after infection or sorted for GFP positivity 48 hours after infection for cell proliferation assay, colony assay, or biochemical experiments.