Ncounter mouse pancancer pathways panel

GSEA was performed on normalized count gene expression data using the GSEA software v4.0.3 of the Broad Institute [20 (link), 21 (link)]. Gene sets were compiled as predefined by NanoString Technologies’ nCounter® (Mouse) PanCancer pathways panel (https://www.nanostring.com/support-documents/ncounter-pancancer-human-pathways-panel-gene-list/?dl=1). Gene sets with a size <15 or >100 genes were excluded from the analysis. Genes were ranked by log2 ratio of classes or diff of classes (for log-scale data) and statistical significance was determined by 1000 gene set permutations. p < 0.05 was considered significant.

Total RNA from sorted CD45⁻ tumor cells was extracted using a QIAshredder kit (QIAGEN) and an RNeasy Plus Micro Kit (QIAGEN) according to the manufacturer’s protocol. RNA quantification was performed using the DeNovix DS-11 Spectrophotometer (DeNovix, Inc). 100 ng of purified RNA was added to 3 μL of Reporter CodeSet and 2 μL Capture ProbeSet using an nCounter master kit as recommended (NanoString Technologies). Samples were processed on the NanoString nCounter Flex System per manufacturer instructions using the nCounter Mouse PanCancer Immune Profiling panel or the nCounter Mouse PanCancer Pathways panel (NanoString Technologies). Each gene set interrogates 750 cancer-related genes alongside 20 internal reference controls (full gene list and controls available on manufacturer’s website). Differentially expressed genes were identified in nSolver 4.0 Analysis Software (NanoString) as genes with a p value of less than 0.05 versus the respective baseline control. Reactome pathway analysis was performed using the NetworkAnalyst. The NanoString data have been deposited in the NCBI GEO under accession number GSE178135.

A minimum of 100 ng RNA per human tissue sample was analyzed using the nCounter PanCancer pathways panel on the nCounter FLEX Analysis System (NanoString Technologies, Seattle, Washington, USA) at the Institute of Pathology of the LMU in Munich.
A minimum of 30 ng RNA per mouse tissue sample was analyzed using the nCounter Mouse PanCancer pathways panel on the nCounter SPRINT Profiler System (NanoString Technologies) at the Nanostring Core Facility of the University Medical Center Hamburg-Eppendorf in Hamburg.
Nanostring gene expression data was compiled, normalized, and log2-transformed using the nSolver Analysis Software 4.0.70 including the nCounter Advanced Analysis Software 2.0.115.