NGS of mus musculus iBMDM CRISPR screen
Cas9-expressing iBMDMs were generated from CRISPR-Cas9 Knockin mice67. A total of 300  106 Cas9-iBMDMs were distributed across 12 of the 15 cm2 tissue culture dishes (25  106 cells per dish) and infected with the Brie library of lentiviral particles at an MOI of 0.3 in 25 ml of complete DMEM (DMEM supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS; S1620, Biowest)) supplemented with 100 l of LentiBOOST transduction reagent (SB-P-LV-101-02, Lentivirus Transduction Enhancer Solution, Siron Biotech) and incubated for 24 h for efficient transduction. These transduced cells were expanded with intermittent passaging to avoid overcrowding of the cells and to generate a sufficient number of cells for the downstream whole-genome CRISPR-KO screens. Two replicates of an adequate number of cells were used as a control to obtain a representation (screen depth) of > 500 cells for each sgRNA of the library, and a similar number of iBMDMs from the same preparation of cells were treated with vehicle control (DMSO) or the TAK1 inhibitor 5z-7-oxozeaenol (5z7) (#17459, Cayman Chemical) at 100 nM final concentration for 24 h. The vehicle-treated control cell population and the surviving cells from the TAK1i-treated samples were subjected to CRISPR screen enrichment analysis.