MCST_Analysis_2026

Morphology Guided Spatial Transcriptomics (MCST) Analysis (Hypertrophic Chondrocytes)

Methods Overview

This repository contains analysis workflows, organized into rendered notebooks for all computational figures, for morphology-guided spatial transcriptomics and single-cell transcriptomic profiling in the hypertrophic chondrocytes for the MCST project. The major analytical components include:

• PIP-seq data processing, integration, clustering, and Slide-seq spot deconvolution
• CurioSeeker spatial transcriptomics processing, TopoVelo velocity analysis and Hypertrophic Chondrocytes analysis
• CytoSignal-based cell-cell communication analysis from spatial transcriptomics data

PiP-seq Processing, LIGER Analysis, And Slide-seq Deconvolution

This section covers single-cell PiP-seq preprocessing, integration of control and knockout datasets, identification of chondrocyte populations, differential analysis in hypertrophic chondrocytes, and RCTD-based deconvolution of Slide-seq spots using the final PiP-seq reference.

Subsections included :

• PIPseeker data QC and filtering
• Doublet detection with DoubletFinder
• Dataset integration and Leiden clustering with rliger
• Chondrocyte marker-based identification and subclustering
• Slide-seq spots deconvolution using PIPseq expression with spacexr
• PIPSeq velocity analysis using veloVAE

Files:

• Figures_2A_S2_PIPSeq_LIGER_analysis.html
• Figure_S3_PIPSeq_velocity_analysis.ipynb

CurioSeeker Spatial Processing And Velocity Analysis

This section summarizes spatial transcriptomics processing for CurioSeeker data, spot-to-cell aggregation, bone-region restriction, and spatio-temporal velocity modeling in the growth plate.

Subsections included :

• Spot-level quality control and normalization
• Cell segmentation based spot aggregation
• Bone-region subsetting and aggregation
• Spatio-temporal velocity inference with TopoVelo
• Hypertrophic chondrocytes
  • Differential Expression Analysis in HCs
  • Cell Area and Transcriptional Density
  • GSEA and GO enrichment analysis

Files:

• Figure_S2-2A_E14SlideSeq_RCTDplotting.ipynb
• Figure_S2-2A_E16SlideSeq_RCTDplotting.ipynb
• Figure_S2-2A_E18SlideSeq_RCTDplotting.ipynb
• Figures_3B_S3-B_E14SlideSeq_TopoVelo.ipynb
• Figures_2C_2D_2E_S2-2D_HCanalysis.ipynb
• Figures_2F_S2-2B_HC_GSEA_GO.html

CytoSignal Cell-Cell Signaling Analysis

This section focuses on spatially resolved cell-cell communication analysis from CurioSeeker expression data, including neighborhood definition, ligand-receptor interaction testing, differential signaling analysis between conditions, and visualization of significant signaling changes.

Subsections included :

• Spot-level QC for signaling analysis
• Contact-dependent interaction analysis
• Diffusion-dependent interaction analysis
• Differential signaling modeling across conditions

Files:

• Figures_4B_4C_S4_CytoSignal_analysis.html