Neutrophil Transcriptome in CB2-Deficient Mice

Wild-type (WT) and CB₂^−/− neutrophils harvested from dorsal air pouches at 6 h post–Zymosan challenge were negatively selected (Miltenyi Biotec, Bergisch Gladbach, Germany), and transcriptome analysis was carried out using the nCounter Mouse Inflammation V2 panel (NanoString Technologies, Seattle, WA, USA) consisting of 248 genes and 14 positive/negative probes. Cells (5 × 10⁶/ml) were lysed and processed according to the manufacturer’s guidelines. Data were analyzed in R (v.3.3.1) using the NanoStringDiff package (v.1.2.0) (27 (link)) and its default settings. Briefly, raw nCounter data were converted into a NanoStringSet object including 6 positive controls, 8 negative controls, and 5 housekeeping genes (Cltc, Gapdh, Gusb, Pgk1, and Tubb5) per sample. The data were normalized and analyzed for differentially expressed genes according to NanoStringDiff instructions following a 2-group comparison approach.