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BU3 NKX2-1GFP hiPSCs were plated onto vitronectin-coated tissue culture plates at 30% confluence one day before transfection. 2 µg of sgRNA and 7 µg donor template were diluted in OptiMEM, mixed at a 1:4 ratio with Lipofectamine STEM (Life Technologies) and then applied to the cells. E8 culture medium containing 5.25 µg/ml Blasticidin was supplied to the cells 24 h after transfection. Selection continued for two days, after which cells were fed fresh E8 medium daily until colonies were large enough for picking and genotyping.
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