Ncounter mouse myeloid innate immunity v2 panel

Total RNA was isolated from purified tumor-infiltrating mouse leukocytes using ReliaPrep™ RNA Cell Miniprep System (Promega) followed by DNAse treatment with DNA-free DNA Removal kit (Thermo Fischer Scientific). Purified total RNA was quantified by Qubit (Thermo Fischer Scientific) and checked for quality by the Bioanalyzer RNA 6000 Nano assay (Agilent Technologies). Gene expression was quantified with the NanoString nCounter platform using 100 ng of total RNA in the nCounter® Mouse Myeloid Innate Immunity panel v2 (NanoString Technologies). Briefly, the code set was hybridized with the purified RNA overnight at 65 °C. RNA transcripts were immobilized and counted using the NanoString nCounter Digital Analyzer. Normalized raw expression data were analysed when two SDs above the geometric mean of the code-set-internal negative control probes were reached. The 557 remaining genes after background filtering were normalized to the geometric mean of 18 housekeeping genes included in the panel and were log2-transformed for further analysis.

Blood was collected and pooled from wild-type or Mvk^VI/Δ91 female mice (n = 4 mice per genotype). PBMCs were isolated by centrifugation over Ficoll-Paque Plus (GE Healthcare) in SepMate tubes (STEMCELL Technologies) and then RNA was extracted using TRI reagent (Sigma-Aldrich, T9424). RNA (50 ng) was hybridized at 65°C overnight according to the manufacturer’s instructions in a thermocycler using the nCounter Mouse Myeloid Innate Immunity Panel V2 (Nanostring Technologies). Hybridized samples were purified and immobilized onto a sample cartridge using the nCounter Prep Station and then analyzed using the nCounter Digital Analyzer. Raw mRNA abundance frequencies were normalized to housekeeping and positive control genes using the nSolver Analysis Software 4.0. Values are expressed as log₂ counts and plotted using the ggplot2 R-package (https://ggplot2.tidyverse.org).