Single-cell spatial transcriptomic profiling defines a pathogenic inflammatory niche in chronic active multiple sclerosis lesions - snRNA-seq

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Frozen autopsy brain tissue from fourteen multiple-sclerosis donors and three neurologically healthy controls was sectioned at –20 °C into 80–100 µm slices, and an 8 mm punch (~25 mg) of white matter or matched control tissue was collected from each region of interest . Nuclei were liberated with the 10x Genomics Chromium Nuclei Isolation Kit, using an extended lysis–debris-removal protocol tailored to lipid-rich white matter; the final suspension was stained with propidium iodide and counted before loading. Single-nucleus libraries were prepared with Chromium 3′-v3.1 reagents and sequenced on an Illumina NovaSeq 6000 to ~30 000 read pairs per nucleus, generating ~20 000 quality-filtered nuclei per sample and 306 731 nuclei in total . Reads were aligned to the GRCh38 reference with CellRanger 7.1.0 ( --include-introns ), and nuclei were retained if they contained 700–25 000 UMIs, 500–7 500 genes and < 5 % mitochondrial transcripts. Per-sample objects were log-normalised in Seurat v5, highly variable genes selected, and batch effects removed with the HarmonyIntegration workflow; clustering was performed in PCA–UMAP space and the cleaned layers were merged with JoinLayers to yield a unified atlas. Finally, CytoSPACE was used to project the transcriptomic clusters onto matched MERFISH sections, assigning each nucleus to demyelinated, perilesional, or healthy white-matter domains and thereby linking cell states to their spatial lesion context.