Ncounter mouse inflammation v2 panel

The analysis of gene expression in the immune response was performed using the NanoString nCounter™ Mouse Inflammation v2 Panel (NanoString Technologies, Inc., Seattle, WA, USA). For sample preparation, total RNA was extracted from RAW264.7 cells after treatment. The quantity and quality of the extracted RNA were measured using a DS-11 spectrophotometer (DeNovix Inc., Wilmington, DE, USA) and an AATI Fragment Analyzer (Agilent Technologies, Santa Clara, CA, USA). The samples with sufficient RNA purity, demonstrated by an RNA concentration of 20 ng/μL or more and an acceptable 260 nm/280 nm absorbance ratio were used. Total RNA was hybridized to a reporter-capture probe at 65 °C for 24 h. After the hybridization reaction, the samples were loaded onto an nCounter cartridge (NCT-120) and the data were gathered by the nCounter Prep Station and the nCounter Digital Analyzer. The raw data were normalized to the housekeeping gene expression and expressed as fold-change. A heatmap was generated using the normalized data.

RNA from whole blood was isolated from Tempus blood RNA tubes according to the manufacturer’s protocol (Tempus Spin RNA Isolation Kit, Thermo Fisher Scientific). Moreover, 50 ng of RNA was hybridized to reporter and capture probe sets of the nCounter Human Immunology v2 panel (Nanostring Technologies) at 65 °C for 24 hours. For in vivo experiments, 100 μL of whole blood collected 1 day postvaccination was lysed with BD PharmLyse reagent. RNA was subsequently extracted with the QIAGEN RNAeasy micro kit. 50ng of RNA was hybridized to the nCounter Mouse Inflammation v2 panel (NanoString Technologies) as previously described [28 (link)]. Unsupervised PCA performed to visualize variability between immunization routes was performed with Partek Genomics Suite Analysis v.7 software. Hierarchical clustering was performed with Seaborn’s Clustermap function in Python version 3.

mRNA was isolated as above from distal colon sections from sham- and DSS-treated (day 8 and day 14) WT and male homozygous Zip8 393T-KI mice. Transcriptomic analysis was performed on the nCounter Mouse Inflammation V2 panel (Nanostring Technologies), consisting of 248 genes and 6 housekeeping genes according to the manufacturer’s protocol. Raw reads were normalized by background thresholding, geometric mean of positive controls, and code set content normalization with housekeeping genes. Data analysis was performed using R (v3.3.2) and NanoString Advanced Analysis platform for differential gene expression.

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.

Lung fibroblasts from early-stage metastatic lungs were sorted and RNA was isolated as described above and analyzed with nCounter Mouse Inflammation v2 Panel, providing gene expression analysis for 248 inflammation-related mouse genes (and 6 internal reference controls), (NanoString Technologies). Raw data were normalized to internal reference genes using the nSolver software, following the manufacturer’s instructions.

Snap-frozen skin was ground to a fine powder in liquid nitrogen using a mortar and pestle. RNA was extracted from the powdered tissue using an RNeasy fibrous tissue mini kit (Qiagen, Germantown, MD, USA). Gene expression was studied using the nCounter Mouse Inflammation V2 Panel (NanoString Technologies, Seattle, WA, USA). Differential gene expression was analyzed by using nSolver software (NanoString Technologies). Bioinformatic analyses for the identification of canonical pathways were performed using Ingenuity Pathway Analysis software (Qiagen).