RNA Extraction and Gene Expression Analysis of Macrophages from Glioblastoma Tumors

RNA from FACS sorted macrophages (CD45^highCD11b⁺) of Gl261 tumors of ICB R and ICB NR mice was extracted using the PicoPureTM RNA Isolation Kit (Arcturus; KIT0202) and gene expression analysis was performed using the nCounter Mouse Immunology Panel (NanoString; XT-CSO-MIM1-12) with the nCounter NanoString™ technology (NanoString Technologies; Seattle, WA)^{57 (link)}. RNA input per sample was 25 ng. Data analysis were performed by nSolver 3.0. Marker for pro- and anti-inflammatory gene signatures were selected according to previously described marker. Gene signature scores were calculated as geometric mean of each gene expression. Controls and low count genes were removed from the NanoString count matrix, followed by a scalar normalization and variance modeling^{58 (link),59 (link)}. Differential gene expression analysis was performed by an eBayes adjusted moderated t-statistic linear regression model^{60 (link)}. Pro- and anti-inflammatory metagene signatures were generated from previously reported markers (Supplementary Table 1) and the geometric mean was estimated in each sample for the different signatures.