NGS- IL-12 treated primary murine neuronal cultures
We performed next generation sequencing of primary murine neurons stimulated with recombinant IL-12 to test the isolated effect of IL-12 on neurons, independent from a heavily inflamed CNS microenvironment. Principal component analysis (PCA) revealed a dose-dependent response to IL-12 in the neuronal transcriptome. Our computational analysis identified 1,878 significantly DEGs (p≤0.01, log ratio ≥0.5). Several of the upregulated genes have been linked to promoting neurogenesis (Fpr1, Mbdi1), neuroprotection (Timp1, Angptl4) and enhancing remyelination (Timp1, Matn2), thereby suggesting a direct neuroprotective and neurotrophic effect of IL-12. Sequencing raw data and processed gene expression data will be deposited into the GEO repository upon publication. Code for analyses will be available upon contacting the corresponding authors: email@example.com and firstname.lastname@example.org Sequencing raw data and processed gene expression data will be deposited into the GEO repository upon publication.
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The raw reads were aligned to mouse genome build GRCm39 using the STAR aligner, with FeatureCounts used to calculate read counts per gene based on GENCODE gene annotation release M26. Bulk RNA-seq analysis was performed using the edgeR and DEseq2 packages. Counts were counts-per-million (CPM) normalized, log2 transformed and only genes were retained if expressed at one CPM in at least two samples. PCA was computed using the BiocGenerics package after variance stabilizing transformation and visualized using ggplot2. Differentially expressed genes between conditions were computed using gene-wise negative binomial generalized linear models and applying a Benjamini-Hochberg correction. Heatmaps of DEGs were generated using pheatmap. Differential analysis of modular gene co-expression was performed using the R implementation of CEMiTool usind default parameters. For gene set enrichment analysis and interactome integration murine gene names have been converted to human orthologs using biomaRt. Gene set enrichment analysis of gene modules was performed using the hallmark gene set collection from MSigDB85. Interactome data from the STRING database version 11 was integrated after filtering on interactions with a minimum score of 20086 to obtain gene co-expression and interaction networks for each module.