Lentiviral gene transfer

The ER-Hoxb8 C57BL6/N conditionally immortalized neutrophil progenitor cell lines were established as described in⁵⁶. In general, 0.3 × 10⁵ C57BL6/N-derived ER-Hoxb8 conditionally immortalized neutrophil progenitors containing Cas9-GFP construct were resuspended in 30 μL of growth medium (RPMI-1640 medium (Corning, 10-040-CV, Corning, NY, USA) supplemented with 10% Fetal Bovine Serum (FBS, Thermo Fisher Scientific, 26140, Waltham, MA, USA), 100 U/ml penicillin/100 μg/mL streptomycin (Thermo Fisher Scientific, 15140-122, Waltham, MA, USA), 2 mM L-Glutamine (Thermo Fisher Scientific, 25030081, Waltham, MA, USA), 2% conditioned CHO media containing Stem Cell Factor (SCF), 0.5 μM β-estradiol (Sigma-Aldrich, E2758, Saint Louis, MO, USA)) and transduced with lentiviral guides (Supplementary Table ²). Control cells were transduced with a guide to knockdown GFP. The neutrophil progenitors were spinfected at 730 g for 1 h at 15 °C in the presence of polybrene (EMD Millipore, TR-1003-G,, Burlington, MA, USA) (8 μg/ml), then 200 μL of fresh culture medium was added to the cells. After 48 h, 5 μg/mL puromycin (Fisher Scientific, ICN19453910, Waltham, MA, USA) were added to the cultures. The puromycin selection was completed within 72 h, after which the cells were transferred to fresh culture media without puromycin, to expand.