Bulk RNA sequencing for sNinj1-mimetic peptide-treated classically activated macrophages

We have applied bulk RNA sequencing as an unbiased transcriptomics to demonstrate the mechanism underlying actions of sNinj1 in macrophage inflammation. The anti-inflammatory role of Ninj1 was verified by treating macrophages with the peptide Ninj11-56 (ML56), which mimic the soluble form of Ninj1 (sNinj1). Peripheral blood samples from 10- to 16-week-old C57BL/6J (B6) mice (n=60, 1 ml per mouse) were collected in EDTA-coated tubes, pooled according to the experimental requirements. The CD11b-selected monocyte suspension was cultured for 6 or 7 days with RPMI/FBS plus 30 ng/ml M-CSF. 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).