GeCKO-screen (DNA-seq) of immortalized ER-Hox8 monocytes
Amplification of mouse CRISPR Knockout pooled library (GeCKO v2) in lentiGuide-Puro plasmid, purchased from AddGene (#1000000053) (Sanjana, Shalem and Zhang, 2014), was performed as described (Joung et al., 2017). Cas9-expressing ER-Hoxb8 cells, transduced with library lentiviral particles at a MOI of 0.4, were differentiated to day 3. Intracellular S100A9 was stained with a S100A9-FITC coupled antibody using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Cells with no/lower S100A9-expression (hits) and cells with normal S100A9-expression (reference) were sorted using a SH800S Cell Sorter (Sony, Minato, Japan) and DNA was purified by phenol-chloroform extraction. Next generation sequencing was performed as described earlier (Joung et al., 2017).