DOI: 10.5061/dryad.15dv41p9d
title: Single-cell spatial transcriptomics of an inducible destabilized-domain Cre mouse line to target disease associated microglia

description: This dataset contains the results from a single-cell spatial
 transcriptomics experiment performed using Bruker Nanostring CosMx
 technology on 10µm thick fresh-frozen coronal brain sections from the
 following groups: 5xFAD hemizygous, Cst7 DD-Cre, Ai14tdTomato
 double heterozygous (AD, n = 3), and Cst7 DD-Cre/Ai14tdTomato
 double heterozygous (WT, n = 1) mice treated with TMP, and Cst7
 DD-Cre/Ai14tdTomato double heterozygous mice treated with cuprizone (CPZ)
 and TMP (CPZ, n = 2). The dataset is provided as an .RDS file,
 which includes raw and corrected counts, along with comprehensive
 metadata. Metadata includes experimental group, sample ID, cell type
 annotations, and X-Y coordinates of each cell. The function of microglia
 during progression of Alzheimer's disease (AD) can be investigated
 using mouse models that enable genetic manipulation of microglial
 subpopulations in a temporal manner. We developed mouse lines that express
 either Cre recombinase (Cre) for constitutive targeting, or
 destabilized-domain Cre recombinase (DD-Cre) for inducible targeting from
 the Cst7 locus (Cst7 DD-Cre) to specifically manipulate disease associated
 microglia (DAM) and crossed with Ai14 tdTomato cre-reporter line mice.
 Cst7Cre was found to target all brain resident myeloid cells, due to
 transient developmental expression of Cst7, but no expression was found in
 the inducible Cst7 DD-Cre mice. Further crossing of this line with 5xFAD
 mice combined with dietary administration of trimethoprim to induce DD-Cre
 activity produces long-term labeling in DAM without evidence of leakiness,
 with tdTomato-expression restricted to cells surrounding plaques. Using
 this model, we found that DAMs are a subset of plaque-associated microglia
 (PAMs) and their transition to DAM increases with age and disease stage.
 Spatial transcriptomic analysis revealed that tdTomato+ cells show higher
 expression of disease and inflammatory genes compared to other microglial
 populations, including non-labeled PAMs. These models allow either
 complete cre-loxP targeting of all brain myeloid cells (Cst7Cre), or
 inducible targeting of DAMs, without leakiness (Cst7 DD-Cre).
description: # Single-cell spatial transcriptomics of an inducible destabilized-domain
 Cre mouse line to target disease associated microglia Dataset DOI:
 [10.5061/dryad.15dv41p9d](https://doi.org/10.5061/dryad.15dv41p9d) ##
 Description of the data and file structure **Sample preparation:**
 Isopentane fresh-frozen brain hemispheres were embedded in OCT compound,
 and 10 μm thick coronal sections prepared on a cryostat. Six hemibrains
 were mounted directly onto a VWR Superfrost Plus microscope slide and
 stored at −80°C until fixation. 5xFAD hemizygous, *Cst7* ^DD-Cre^,
 Ai14^tdTomato^ double heterozygous (*n* = 3), and *Cst7*
 ^DD-Cre^/Ai14^tdTomato^ double heterozygous (*n* = 1) mice treated with
 TMP, and *Cst7* ^DD-Cre^/Ai14^tdTomato^ double heterozygous mice treated
 with cuprizone (CPZ) and TMP (*n* = 2) were used for spatial
 transcriptomics. Tissues were processed according to the Nanostring CosMx
 fresh-frozen slide preparation manual for RNA assays. **Data processing:**
 Spatial transcriptomics datasets were filtered using the AtoMx RNA Quality
 Control module to flag poorly performing probes, cells, FOVs, and target
 genes. Datasets were then normalized and scaled using Seurat SCTransform
 to account for differences in library size across cell types (Hafemeister
 and
 Satija [2019](https://onlinelibrary.wiley.com/doi/10.1002/glia.70004#glia70004-bib-0019); Choudhary and Satija [2022](https://onlinelibrary.wiley.com/doi/10.1002/glia.70004#glia70004-bib-0008)). Principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) analysis were performed to reduce dimensionality for downstream analysis. Unsupervised clustering at 1.0 resolution yielded 38 clusters for the dataset. Clusters were manually annotated based on gene expression and spatial location. We have submitted the processed .RDS file, analyzed using the R package Seurat. Sample metadata are stored in `annotated_seurat@meta.data`. ### Files and variables **annotated_seurat.RDS** Row names of metadata (accessed using rownames(`seurat@meta.data`)) contain unique identifiers for each single cell, formatted as `c_[slide]_[fov]_[cell]`. Cell type annotations are stored in Idents(seurat). 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 DAPI fluorescence intensity within a given cell (AU) * Max.DAPI: Max DAPI fluorescence intensity within a given cell (AU) * Mean.Histone: Mean Histone fluorescence intensity * Max.Histone: Max Histone fluorescence intensity * Mean.rRNA: Mean rRNA fluorescence intensity * Max.rRNA: Max rRNA fluorescence intensity * Mean.GFAP: Mean GFAP fluorescence intensity * Max.GFAP: Max GFAP fluorescence intensity * 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 * nCount_SCT: SCT-corrected number of RNA counts * nFeature_SCT: SCT-corrected number of unique RNA targets * nFeature_negprobes: Number of unique Negative targets * Area.um2: Area of cell (um^2) * qcCellsFlagged: Cells flagged by AtoMx QC module * group: Experimental group name * sample_n: Sample number ## Code/software Files were analyzed using Seurat ([https://satijalab.org/seurat/](https://satijalab.org/seurat/)).