Dysbiosis alters the mRNA transcript profile in both spleen and MLNs. RNA was isolated from whole-cell isolates of the spleen and MLNs of ampicillin-treated compared to control mice at 6 weeks of age, before FVIII infusion (n = 3 in each). RNA was analyzed using a Nanostring murine immunology panel of 547 genes. (A) Differences in gene expression within the spleen (left) and MLNs (right) as a result of ampicillin-treatment is visualized on a volcano plot. An arbitrary fold-change cutoff of ±1.25 and an α = 0.05 was applied to focus on potentially significant genes, and those that met criteria are highlighted in red. Significance was calculated using the Welch t test for unequal variances. (B) Differences in gene expression between both cohorts are represented through their respective z-scores on a heat map. Clustering was completed by using the Pearson squared distance metric of z-scores with Ward’s clustering method.