Optimized nucleofection protocol for preMacs with subsequent microglia differentiation

25 pmol of sgRNA was complexed with 2 µg of TrueCut Cas9 protein v2 (Invitrogen) for 20 min at room temperature. 50,000 preMacs per reaction were resuspended in 20 µL P3 Primary Cell Nucleofector solution (Lonza) and mixed with CRISPR-Cas9 RNPs before nucleofection with CM-137 pulse code using a 4D-Nucleofector 96-well Shuttle nucleofector (Lonza). After nucleofection, cells were allowed to rest for 5 min before 80 µL microglia differentiation medium (see earlier text) was added per well and suspension was transferred to a fibronectin-coated seeding plate (PhenoPlate, PerkinElmer) containing 100 µL differentiation medium. 75% of the medium was changed the day post-nucleofection, and then 50% medium changes were carried out every two days.