Spatial Transcriptomics Data and Computational Tools for Integrated Super-Resolution In Situ Sequencing and Protein Imaging for 3D Characterization of Intact Human Brain Organoids

This repository provides the complete set of data and analysis scripts supporting the study titled:“Integrated Super-Resolution In Situ Sequencing and Protein Imaging for 3D Characterization of Intact Human Brain Organoids” The dataset includes processed CSV files containing 3D spatial coordinates (x, y, z) and gene identities for individual RNA puncta detected in expanded human brain organoids. These organoids were generated from induced pluripotent stem cells (iPSCs) derived from patients carrying the iSTXBP1/PTHS mutation and their sex-matched healthy family members. The data were acquired using two complementary techniques: Expansion Sequencing (ExSeq): A super-resolution, in situ RNA sequencing method that allows nanoscale localization of transcripts within intact, physically expanded organoids. Ex situ RNA sequencing: Conventional transcriptomic profiling performed outside the tissue context to provide additional validation and complementary expression data. Together, these methods enabled a high-resolution, spatially informed characterization of gene expression patterns in disease-relevant and control brain organoids. In addition to the spatial transcriptomics data, the repository includes Python and MATLAB scripts designed for quantitative spatial analysis and visualization. These scripts perform: 3D segmentation of nuclei or cell boundaries, Distance-based analyses (e.g., distances to centroids or edges), Spatial statistics such as Moran’s I for assessing local spatial autocorrelation, The combination of ExSeq and Ex situ data, along with this computational toolkit, allows researchers to explore the spatial organization of gene expression at subcellular resolution and identify potential spatial signatures associated with neurodevelopmental disorders. Please refer to the included README.md file for detailed descriptions of file structure, dependencies, and analysis steps.