Spatial Regulation of Protocadherins in Mouse Cortex

MERFISH-based spatial transcriptomic analysis of protocadherin gene expression in mouse cortex (WT vs KO Mouse Cortex)

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Overview

This repository contains analysis pipelines for MERFISH spatial transcriptomics data comparing WT vs KO in mouse cortex.

The workflow includes:

• MERFISH data QC and preprocessing
• Clustering gene expression and cell type annotation
• Cortical layer-wise analysis
• Protocadherin (Pcdh) expression and similarity analysis

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Dependencies

These analyses were performed using:

R environment

• R version: 4.4.1
• Platform: x86_64-w64-mingw32 (Windows 11 Pro)

Python environment

• Ubuntu 22.04.5 LTS
• For installation:

conda create -n STenv python=3.10 scanpy=1.10.1 squidpy=1.2.2 -c conda-forge -y
conda activate STenv

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Main R packages

Seurat_5.4.0, SeuratObject_5.3.0, harmony, dplyr_1.1.4, ggplot2_4.0.2, patchwork_1.3.2, Matrix_1.7-1, FNN_1.1.4.1, SeuratWrappers_0.3.2

• For installation

install.packages("remotes")
remotes::install_version("Seurat", version = "5.4.0")
remotes::install_version("SeuratObject", version = "5.3.0")
remotes::install_version("dplyr", version = "1.1.4")
remotes::install_version("ggplot2", version = "4.0.2")
remotes::install_version("patchwork", version = "1.3.2")
remotes::install_version("Matrix", version = "1.7-1")
remotes::install_version("FNN", version = "1.1.4.1")
install.packages("harmony")
remotes::install_github("satijalab/seurat-wrappers@v0.3.2")

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Other tools

• locator() (base R) for manual tracing along the cortical layers

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Main Scripts

QC_&_filtering.py
: Quality control and filtering of MERFISH data

integrate_&_get_DEG_removed_clusters.R
: Normalization, integration (Harmony), clustering, and UMAP generation

get_celltype_annotations.R
: Cell type marker-based annotation of clusters

get_cortical_layer_anchor_points.R
: Manual cortical layer tracing and anchor point generation

anchor_neighborhood_cells_#Pcdh_genes_expressed.R
: Extraction of neighboring cells and number of Pcdh genes expressed in neighborhood cells

anchor_neighborhood_cells_get_Jaccard_index_for_Pcdh.R
: Binarized Pcdh gene expression similarity analysis (Jaccard index) in neighborhood cells

anchor_neighborhood_cells_Pcdh_Spearman_correlation.R
: Spearman correlation of Pcdh gene expression in neighborhood cells

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Data Access

The dataset is available upon request to the reviewers

Contact

For questions, please contact:
Pubudu Kumarage (pkumarage@wisc.edu), Yu Gao (yu.gao@wisc.edu), Xinyu Zhao (xinyu.zhao@wisc.edu)