Single-cell spatial transcriptomics of ACAN cKO in WT and 5xFAD mice
Data files
Jul 01, 2025 version files 3.96 GB
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5xACANcKO_RNA_slide1.rds
2.14 GB
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5xACANcKO_RNA_slide2.rds
1.83 GB
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README.md
2.13 KB
Abstract
This dataset comprises results from one single-cell spatial experiment conducted on mouse brains. This experiment was performed using the Bruker Nanostring CosMx technology on 10 µm coronal brain sections from 8-month-old female WT, WT ACAN cKO, 5xFAD, and 5x ACAN cKO mice. The dataset is provided as two separate RDS files split by flowcell which include raw and corrected counts for the RNA data, along with comprehensive metadata. Metadata includes mouse genotype, sample ID, cell type annotations, and X-Y coordinates of each cell.
Dataset DOI: 10.5061/dryad.z612jm6pw
Description of the data and file structure
Due to the large file size, the R object has been split by flowcell into two separate files (5xACANcKO_RNA_slide1.rds and 5xACANcKO_RNA_slide2.rds). The two files should be loaded into the R workspace and combined using the merge() function. For merging and downstream analysis, we recommend using a high performance computing system and at least 64GB of RAM for optimal performance. Data were analyzed using the R package Seurat. Sample metadata are stored in seurat@meta.data.
Files and variables
Single-cell spatial transcriptomics dataset
Rownames of metadata (accessed using rownames(seurat@meta.data) contain unique identifiers for each single cell, formatted as c_[slide][fov][cell]. Additional metadata columns are described below:
- fov: Field Of View (FOV) the cell is in
- Area: Number of pixels assigned to a given cell
- AspectRatio: Width divided by height
- x_FOV_px: x position of the cell center within the FOV, measured in pixels
- y_FOV_px: y position of the cell center within the FOV, measured in pixels
- Width: Cell’s maximum length in x dimension (pixels)
- Height: Cell’s maximum length in y dimension (pixels)
- Mean.DAPI: Mean fluorescence intensity within a given cell (AU)
- Max.DAPI: Max fluorescence intensity within a given cell (AU)
- Run_Tissue_name: Flowcell name
- slide_ID_numeric: SlideID
- x_slide_mm: x position of the cell center within the slide, measured in mm
- y_slide_mm: y position of the cell center within the slide, measured in mm
- nCount_RNA: Number of RNA counts
- nFeature_RNA: Number of unique RNA targets
- nCount_negprobes: Number of Negative counts
- nFeature_negprobes: Number of unique Negative targets
- Area.um2: Area of cell (um^2)
- reannotation: Manual cell type annotation based on marker genes and location in space
- reannotation_broad: Broad cell type annotation (e.g., all excitatory neurons grouped together)
- group: Genotype name
- sample_n: Sample name
Sample preparation: Isopentane fresh-frozen brain hemispheres were embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura Fintek, Torrance, CA), and 10 µm thick coronal sections were prepared using a cryostat (CM1950, LeicaBiosystems, Deer Park, IL). Six hemibrains were mounted onto each VWR Superfrost Plus microscope slide (Avantor, 48311-703) and kept at -80°C until fixation. For transcriptomic analysis, n=3 mice per genotype were used for both the 5xFAD and 5xFAD ACAN cKO groups, while n=4 for WT and n=2 for WT ACAN cKO. Tissues were processed according to the Nanostring CosMx fresh-frozen slide preparation manual for RNA assay (NanoString University).
Data processing: Spatial transcriptomics datasets were filtered using the AtoMx RNA Quality Control module to flag outlier negative probes (control probes targeting non-existent sequences to quantify non-specific hybridization), lowly-expressing cells, FOVs, and target genes. Datasets were then normalized and scaled using Seurat 5.0.1 SCTransform to account for differences in library size across cell types. Principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) analysis were performed to reduce dimensionality and visualize clusters in space. Unsupervised clustering at 1.0 resolution yielded 38 clusters. Clusters were manually annotated based on gene expression and spatial location.