# RNA-seq Analysis

This section of the repository contains the R script used to analyze bulk RNA-seq data generated in the study:

**Conti et al., 2025**  
*Senescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6 mediated gene editing in hematopoietic stem cells*.  
Published in **Cell Reports Medicine**.

## 🔬 Study Context

Bulk RNA-seq was used to investigate transcriptional changes in human hematopoietic stem and progenitor cells (HSPCs) following CRISPR-Cas9/AAV6-mediated gene editing. The goal was to assess activation of DNA damage response (DDR), senescence, and inflammatory pathways.

## 📂 Repository Content

- `Conti_GE_2025_RNAseq_plots.R`:  
  The R script used to generate the GSEA-based figures in the manuscript. It includes:
  - Differential enrichment analysis of senescence and SASP-related gene sets
  - Hallmark pathway analysis (MSigDB)
  - Data integration from multiple timepoints (24h and 96h)
  - ggplot-based heatmaps and tile plots for NES and p-value visualization

The script assumes that input GSEA result files are organized in the following directories:
- `paired_stranded24h/` — results from 24h timepoint  
- `unstrand_96h/` — results from 96h timepoint

## 📊 Data Availability

The RNA-seq data are publicly available on the Gene Expression Omnibus (GEO) under the SuperSeries accession **[GSE244257](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE244257)**.

- `GSE244247`: RNA-seq from day 1 post-editing  
- `GSE244248`: RNA-seq from day 4 post-editing

## 📌 Citation

If you use this dataset or code, please cite:

> Conti A., Giannetti K., Midena F., et al. (2025).  
> *Senescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6 mediated gene editing in hematopoietic stem cells*.  
> **Cell Reports Medicine**.  
> GEO accession: GSE244257