🥦 Broccoli Single-Cell RNA-seq Analysis Pipeline

This repository contains a complete pipeline for analyzing single-cell RNA-seq (scRNA-seq) data in broccoli (Brassica oleracea) using the Seurat package for clustering and Monocle 3 for pseudotime/trajectory analysis.

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📌 Project Goal

The aim is to investigate gene expression heterogeneity and senescence progression in broccoli at the single-cell level, particularly focusing on preharvest and postharvest conditions. This pipeline enables:

• Quality control and filtering
• Normalization and dimensionality reduction
• Cell clustering and UMAP visualization
• Marker gene identification
• Pseudotime trajectory analysis

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🧪 Dataset

The input dataset is generated using 10X Genomics single-nucleus RNA-seq (snRNA-seq) on broccoli inflorescence tissue. It includes the following files typically found in the filtered_feature_bc_matrix directory:

matrix.mtx  
barcodes.tsv.gz  
features.tsv.gz  

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🔧 Requirements

Install the required R packages:

install.packages("Matrix")
install.packages("Seurat")
if (!requireNamespace("BiocManager", quietly = TRUE)) install.packages("BiocManager")
BiocManager::install("monocle3")

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🚀 How to Run

Clone this repository:

git clone https://github.com/your-username/broccoli-scRNA-seq.git
cd broccoli-scRNA-seq

Open and run the pipeline in R:

source("broccoli_seurat_pipeline.R")

Make sure to update the setwd() path in the script to where your data files are located.

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📊 Output Files

• cluster_markers.csv: Top marker genes per cluster
• pseudotime_values.csv: Pseudotime values for each cell

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📈 Visualizations Generated

• Violin plots of QC metrics (nFeature_RNA, nCount_RNA)
• PCA and Elbow plots
• UMAP with clusters
• Top variable features plot
• Pseudotime trajectory plot

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🧬 Biological Application

This pipeline is designed to identify senescence-associated genes (SAGs) and understand cell type-specific responses during postharvest senescence. By integrating pseudotime analysis, we aim to reconstruct the senescence trajectory and reveal regulatory mechanisms underlying broccoli quality decline after harvest.