Tpex RNA-seq Analysis

Author: Dr. Iman Sadeghi Dehcheshmeh
Repository: Tpex_RNAseq
Last Updated: October 2025

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🧬 Project Overview

This repository contains a complete analysis workflow for RNA-seq profiling of Tpex cells under AZM (Azithromycin) vs DMSO treatment.
The project integrates differential expression, weighted gene co-expression network analysis (WGCNA), and multi-database functional enrichment (GO, Reactome, KEGG, WikiPathways, and MSigDB Hallmark).

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🧠 Biological Goal

To identify transcriptional modules and gene networks modulated by Azithromycin treatment, with emphasis on:

• Immune regulation and T-cell exhaustion (Tpex-related genes)
• Translational and metabolic reprogramming
• Network hubs and functional pathways driving phenotypic response

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📁 Repository Structure

Tpex_RNAseq/
├── data/
├── results/
│   ├── wgcna_allgenes/
│   ├── enrich_gprofiler/
│   ├── deg_analysis/
└── codes/

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🧩 Key Analyses

Preprocessing

• Top 8 000 genes by MAD
• Filtered for non-zero variance and quality

Differential Expression

• Model: ~ Condition + Donor
• Cutoffs: |log2FC| > 0.5, p < 0.05

Weighted Gene Co-Expression Network

• Power selection via scale-free topology
• Module–trait correlation (AZM vs DMSO, Donor)
• HubScore = z(|kMEₒwn| + |GS| + kWithin)

Functional Enrichment

• g:Profiler: GO, Reactome, KEGG, WikiPathways
• Hallmark: MSigDB H collection
• Integrated Up/Down dotplots (PDF)

Network Visualization

• Cytoscape exports for per-module networks
• Global hub graph (|bicor| ≥ 0.8)

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⚙️ Dependencies

Package	Version
WGCNA	≥ 1.73
limma, DESeq2	≥ 3.56
clusterProfiler, org.Hs.eg.db	≥ 4.8
gprofiler2	≥ 0.2
msigdbr	≥ 7.5
igraph	≥ 2.0
ggplot2, ggrepel, pheatmap	≥ 3.4

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▶️ How to Run

git clone https://github.com/isadeghi87/Tpex_RNAseq.git
cd Tpex_RNAseq/codes

Rscript st1_preprocessing.R
Rscript st2_differential_expression.R
Rscript st3_wgcna_network.R
Rscript st4_enrichment_analysis.R
Rscript st5_visualization.R

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🧠 Summary

• Blue module : immune/lysosomal upregulation by AZM
• Green module : translational downregulation
• Hub genes : IL7R, CXCR4, TSC22D3, FOXP3
• Hallmarks : IL2-STAT5 signaling, apoptosis, oxidative stress

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🛠️ License

MIT License — see LICENSE

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