DOI: 10.5281/zenodo.14988055
title: Visium Spatial and snRNA data of Brain section from Parkinson Mouse Model based on inducible expression of human a-syn constructs: 20-months + snRNA 23 months dataset

description: Using 23-months old mice of a inducible expression of human a-syn constructs based Parkinson mouse model, we produced a single nucleus RNA dataset by cutting 0mm Bregma to -5mm Bregma. The Chromium 3’ Single Cell Library Kit (10x Genomics) was used and Sequencing was performed on a NovaSeq 6000. From the same model we also used 20-months old mice with the Visium Spatial V1 platform (10x Genomics). Sequencing was performed on a NovaSeq 6000. Both were PE150.

snRNA pipeline: For the alignment of reads, a custom reference was created by adding the sequences of the V1S/SV2 transgene and the Camk2a promoter to the mm10 mouse reference genome. Count matrices generated by cellranger count 7.1 were loaded into an AnnData object and processed using the Python-based framework Scanpy 1.10.2. Integration with R, where needed, was facilitated through the rpy2 package. Raw count matrices were corrected for ambient RNA contamination using the SoupX 1.6.2. To remove potential doublets, scDblFinder 1.18.0 was employed with a fixed seed (123). Nuclei with nUMI and nGenes values exceeding three median absolute deviations (MADs) from the median were excluded. Genes detected in fewer than five nuclei across the dataset were excluded. The resulting dataset was normalized via scanpy.pp.normalize_total and scanpy.pp.log1p. Highly variable genes were identified using the function scanpy.pp.highly_variable_genes with the Seurat v3 flavor, selecting the top 4,000 genes. Dimensionality reduction was performed using principal component analysis (PCA) and batch effects were corrected using the python-implemented version of Harmony via the function scanpy.external.pp.harmony_integrate. Harmony embeddings were then used to construct a k-nearest neighbor (kNN) graph with scanpy.pp.neighbors. Clustering was performed using Leiden clustering with standard parameters via the function scanpy.tl.leiden. Clusters were annotated using literature, the mousebrain.org, and markers identified via the FindConservedMarkers function in Seurat. First, neurons and non-neuronal cells were distinguished using mainly canonical markers, such as but not limited to Rbfox3 (neurons), Mbp (oligodendrocytes), Acsbg1 (astrocytes), Pdgfra (oligodendrocyte precursor cells), Inpp5d (microglia), Colec12 (vascular cells), and Ttr (choroid plexus cells). Neurons were further classified into Vglut1 (Slc17a7), Vglut2 (Slc17a6), GABA (Gad2), cholinergic (Scube1), and dopaminergic (Th) neurons. Vglut1 and GABA neurons were further annotated into subtypes based on subclustering and FindConservedMarkers markers. 

visium spatial pipeline: Sequences were fiducially aligned to spots using Loupe Browser ver. 8. All aligned sequences were mapped using spaceranger count 3.0.1 with a custom refence, which included sequences for the promotor and transgene (Camk2aTTA, V1S/SV2) to the mouse genome mm39. We filtered each sample of the Visium Spatial dataset based on the MAD filtering of number of reads (nUMI), number of genes (nGene), and percentage of mitochondrial genes (percent.mt). A spot was filtered out if it was outside of 3x MAD value in at least two metrics. Filtered samples were merged into one Seurat 5.1.0 object and we obtained normalized counts by the SCTransform function of Seurat. Integration was performed using Harmony 1.2.0 on 50 PCA embeddings and clustering was done using Leiden clustering based on 30 harmony embeddings. Integrated clusters were visualized using the UMAP method. Samples that were not successfully integrated (based on similarity measures of the harmony embeddings) and showed high percentage.mt or low nUMI levels compared to other samples, were removed from subsequent analysis. A final integration and clustering were performed after filtering. Regions were first annotated based on a 0.1 resolution clustering to get high level region annotation (Cortex, Hippocampus, Subcortex). Each high-level region was further annotated based on either more granular resolutions or subclustering. Marker genes from mousebrain.org and literature were used in combination with the Allen mouse brain atlas to obtain anatomically relevant annotations.