scRNAseq Analysis Code — Benamar et al.

This repository contains all the code used in the analysis for the manuscript:

"Notch3+ Regulatory T cells Drive Autoimmune Neuroinflammation in Multiple Sclerosis"
Benamar et al.

• Manuscript: [Link] (DOI: [..])
• Contact: Dr. Mehdi Benamar — Mehdi.Benamar@childrens.harvard.edu

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Overview

This repository collects the scripts for processing and analyzing single-cell RNA sequencing (scRNAseq) data as described in the manuscript.
Preliminary analyses were performed using Cellranger (10x Genomics). The analysis workflow was performed using Seurat v4, with additional supporting R packages.

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Analysis Workflow

1. Quality Control and Processing (Seurat)

   • See: script/1_QC_and_processing.R
   • Import filtered matrices from CellRanger, quality control, filtering, normalization, integration, and clustering.

2. Differential Gene Expression (Seurat)

   • See: script/2_DE_genes_with pseudobulk.R
   • Identification of differentially expressed genes using pseudobulk analysis.

3. Visualization and Figure Generation (Seurat)

   • See: script/3_Plots.R
   • Generation of all figures and plots included in the manuscript.

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Data Availability

• Raw sequencing data: Available at GEO/SRA: GSE253602
• Processed Seurat object: Available on Zenodo (DOI: 10.5281/zenodo.15747708)

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Requirements

All R package dependencies and the R version used for this analysis are specified in the renv.lock file included in this repository.

To recreate the exact software environment, install the renv package in R and run:

install.packages("renv")
renv::restore()

This will install the correct R packages and versions as used for the original analysis.

Note:
The analysis was performed using R version 4.3.2
For details about all package versions, please refer to the renv.lock file.

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Usage

1. Clone the repository:

   git clone https://github.com/tuo-utente/nome-repo.git
   cd nome-repo

2. Follow the scripts in the order described above.
   Please refer to comments within each script for details on requirements and parameters.

Note:
For Cellranger usage, refer to the 10x Genomics documentation.

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Issues & Contact

If you encounter any problems or have questions, please open an issue in this repository or contact Dr. Mehdi Benamar at Mehdi.Benamar@childrens.harvard.edu and Dr. Olga Lanzetta at olga.lanzetta@edu.unige.it

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