7  Differential Expression Analysis III

Cre-AAV vs. GFP control in switch mice (batch 2 only)

Published

October 29, 2025

7.1 Pre-filtering low count genes

Genes with very low read counts were excluded to minimize technical noise. After filtering for a minimum of 20 counts in at least five samples, approximately 19011 genes were retained for downstream analysis.

7.2 Differential Expression Analysis: Cre-AAV vs. GFP control in switch mice (batch 2 only)

To avoid statistical complications from cross-batch replicates and batch-treatment nesting, we analyzed only Batch 2 samples (~ Sex + group). This approach ensures true biological independence (each mouse measured once), eliminates batch confounding (both groups processed simultaneously), and provides the cleanest treatment contrast without need for batch adjustment or averaging

7.2.1 Liver

There are 28 significantly differentially expressed genes (FDR < 0.01, |log2FC| > 1) in the liver between Cre-AAV and GFP control switch mice.

Diagnostic plots for the DE analysis

  • A histogram enriched for low p-values shows the presence of true differential expression (deviation from the uniform null), rather than an all-noise result.
  • The log(baseMean) distribution for significant genes being skewed toward moderate–high counts indicates findings are driven by well-expressed features, not low-count noise that can inflate fold-changes and p-values.
Significantly differentially expressed genes in liver
Cre-AAV and GFP control
(FDR < 0.01, |log2FC| > 1)
gene baseMean log2FoldChange padj
Rgs1 277.42 2.58 0.00
ENSMUSG00000140143 112.21 2.12 0.00
Slc41a2 283.20 -1.91 0.00
Mov10 216.60 -1.27 0.00
2810001A02Rik 264.20 1.25 0.00
Ptp4a1 270.44 1.35 0.00
ENSMUSG00000137723 198.27 1.36 0.00
Zfp969 127.80 9.48 0.00
Gm16573 737.60 1.34 0.00
Snhg3 265.15 1.66 0.00
Fasn 1476.70 -1.36 0.00
Rgs4 62.96 1.28 0.00
Fmo3 3565.27 1.35 0.00
Gm45507 260.41 1.27 0.00
Cebpd 337.90 1.04 0.00
Lncbate1 106.93 1.33 0.00
Slc36a1 427.98 -1.06 0.00
Hcn3 76.63 -1.82 0.00
Dpy19l3 125.47 -1.67 0.00
Gm28857 73.70 2.14 0.00
Slc25a47 30280.87 1.03 0.00
E030018B13Rik 63.31 4.72 0.00
Pzp 54787.16 -1.08 0.01
Gm48199 161.83 3.53 0.01
Defb1 146.20 1.31 0.01
Abca2 461.38 -1.05 0.01
Ccdc148 103.67 1.06 0.01
Klhdc7a 477.64 -1.40 0.01

To focus on high-confidence, biologically interpretable results, we applied an additional filtering step to remove pseudogenes (-ps, -rs), unannotated loci (Gm-, Rik, ENSMUS), mitochondrial (-mt), and ribosomal (-Rp) genes. This yielded 20 high-confidence nuclear genes that were consistently dysregulated in response to Cre-mediated ApoE4→ApoE2 conversion in liver, after adjusting for both sex and batch effects.

gene baseMean log2FoldChange padj
Rgs1 277.42 2.58 0.00
Slc41a2 283.20 -1.91 0.00
Mov10 216.60 -1.27 0.00
Ptp4a1 270.44 1.35 0.00
Zfp969 127.80 9.48 0.00
Snhg3 265.15 1.66 0.00
Fasn 1476.70 -1.36 0.00
Rgs4 62.96 1.28 0.00
Fmo3 3565.27 1.35 0.00
Cebpd 337.90 1.04 0.00
Lncbate1 106.93 1.33 0.00
Slc36a1 427.98 -1.06 0.00
Hcn3 76.63 -1.82 0.00
Dpy19l3 125.47 -1.67 0.00
Slc25a47 30280.87 1.03 0.00
Pzp 54787.16 -1.08 0.01
Defb1 146.20 1.31 0.01
Abca2 461.38 -1.05 0.01
Ccdc148 103.67 1.06 0.01
Klhdc7a 477.64 -1.40 0.01

7.2.2 Brain

There are 0 significantly differentially expressed genes (FDR < 0.01, |log2FC| > 1) in the brain between Cre-AAV and GFP control switch mice.

Diagnostic plots for the DE analysis

  • The distribution of p-values from the Wald test was approximately uniform, with no enrichment of low p-values