GSE290106_analysed_experiment_card.md

analysis_date	h5ad_file	species	total_cells	design_type	edviz_grammar	factors	tool_version
2025-11-12	GSE290106_analysed.h5ad	Mouse (Mus musculus)	21417	2×2 Nested Design (Hierarchical)	Genotype(2) > Sample[5103|3881|7904|4537] : CellType(6)	genotype sample cell_type	0.1.0

Experimental Design Card

Dataset Information

File: GSE290106_analysed.h5ad Analysis Date: 2025-11-12 Species: Mouse (Mus Musculus) Total Cells: 21,417

Experimental Context

Experiment Type: Tumor microenvironment profiling study

Research Question: How does Keap1 knockout affect tumor microenvironment composition and immune cell infiltration compared to wild-type?

Factor Descriptions:

• Genotype: Two experimental conditions: KeapKO (Keap1 knockout - constitutive Nrf2 activation and altered oxidative stress response) and KeapWT (wild-type control)
• Sample: Biological replicates (2 per genotype), tumor tissue samples
• Cell Type: Six cell populations identified in tumor microenvironment: CAF (cancer-associated fibroblasts), DC (dendritic cells), Macrophages (dominant population at 92%), NK cells, Neutrophils, and Tumor cells

Design Structure

Identified Factors

Factor	Levels	Type
Genotype	2	Treatment
Sample	4	Replicate
Cell Type	6	Observation

Design Classification

This dataset exhibits a nested design. The factor sample is nested within genotype, meaning each sample belongs to exactly one genotype condition. Cell Types are observed across all samples, creating a crossed relationship with the nested structure.

Design Diagram

┌──────────────────── Design Structure ────────────────────┐
│                                                          │
│ Genotype(2)                                              │
│    ↓                                                     │
│ Sample([5103 | 3881 | 7904 | 4537])                      │
│    :                                                     │
│                                                          │
│ CellType(6)                                              │
│                                                          │
│                                                          │
│                                                          │
│                                                          │
│                                                          │
│                                                          │
└──────────────────────────────────────────────────────────┘

Grammar Notation

Genotype(2) > Sample[5103|3881|7904|4537] : CellType(6)

Distribution Summary

Samples per Genotype: 8,982 - 12,442 (mean: 10,712)
Cells per Sample: 3,881 - 7,904 (mean: 5,356)
Cells per Cell Type: 32 - 19,789 (mean: 3,573)

Analysis Considerations

This design structure has implications for statistical analysis:

Random Effects Modeling: The nesting of sample within genotype indicates that sample-specific variation should be modeled as a random effect. When testing for genotype effects, use mixed-effects models with random intercepts for sample (e.g., ~ genotype + (1|sample) in lme4 notation).

Aggregation Strategy: For differential expression testing, pseudobulking to the sample level preserves the experimental unit structure. Aggregate cells to sample-by-cell_type pseudobulk profiles before applying standard DE methods, treating samples as biological replicates.

Contrast Specification: When comparing genotypes, ensure contrasts are computed at the sample level, not the cell level, to avoid pseudoreplication and inflated Type I error rates.

Design Notes

Highly unbalanced cell type distribution with Macrophages comprising 92.4% of all cells

Unbalanced sample sizes ranging from 3,881 to 7,904 cells per sample

KeapWT samples have more cells overall (12,442) compared to KeapKO (8,982)