Bulk RNA sequencing for classically activated macrophages (CAMs) from Ninj1+/+ and Ninj1-/- mice

We have applied bulk RNA sequencing as an unbiased transcriptomics to explore the association between Ninj1 deletion and macrophage activation. BM cells were isolated from the femurs and tibias of Ninj1+/+ and Ninj1-/- mice at 6 weeks of age and cultured in DMEM containing 20% FBS and 20 μM β-mercaptoethanol for 16 hours. The suspended cells were plated and treated with 20 ng/ml recombinant human/mouse macrophage colony-stimulating factor (M-CSF, 30ng/ml) for 7 days to differentiate macrophages. For activation in vitro, mouse differentiated macrophages were stimulated with lipopolysaccharide (LPS, 500 ng/ml) and IFNγ (20 ng/ml) to M(LPS/IFNγ)s, which were classically activated macrophages (CAMs).