preMac differentiation into microglia and macrophages
For microglia differentiation, preMacs were seeded on fibronectin- coated plates (Corning) in DMEM/F12 + GlutaMAX supplied with N2 supplement (Gibco) and containing human recombinant M-CSF (25 ng/mL), IL-34 (100 ng/mL; Miltenyi Biotec), and transforming growth factor b1 (50 ng/mL; PreproTech). 50% medium was changed every second day for 10 days. Conversely, macrophages were initially cultured without coating in X-VIVO15 medium or ImmunoCult for macrophage differentiation (STEMCELL Technologies) containing 100 ng/mL M-CSF for 6 days, with a 50% medium change on day 3. For nucleofection, macrophages were differentiated in RPMI 1640 with 10% FCS (Gibco) containing M-CSF (100 ng/mL) instead for 6 days.

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.