Single-nucleus RNA sequencing reveals glial cell type-specific responses to ischemic stroke in male rodents

Neuroglia critically shape the brain´s response to ischemic stroke. However, their phenotypic heterogeneity impedes a holistic understanding of the cellular composition of the early ischemic lesion. Here we present a single cell resolution transcriptomics dataset of the brain´s acute response to infarction. Oligodendrocyte lineage cells and astrocytes range among the most transcriptionally perturbed populations and exhibit infarction- and subtype-specific molecular signatures. Specifically, we find infarction restricted proliferating oligodendrocyte precursor cells (OPCs), mature oligodendrocytes and reactive astrocytes, exhibiting transcriptional commonalities in response to ischemic injury. OPCs and reactive astrocytes are involved in a shared immuno-glial cross talk with stroke-specific myeloid cells. Within the perilesional zone, osteopontin positive myeloid cells accumulate in close proximity to CD44+ proliferating OPCs and reactive astrocytes. In vitro, osteopontin increases the migratory capacity of OPCs. Collectively, our study highlights molecular cross talk events which might govern the cellular composition of acutely infarcted brain tissue.

File Name: Supplementary Data 2 Description: Signature gene sets derived from previous studies.This data file contains all gene set lists used for the comparisons of DEGs derived from this dataset with previously published gene sets and the calculation of module scores.
File Name: Supplementary Data 3 Description: Results of differentially expressed gene calculations, for each major cell cluster.This data file contains all DEG counts and non-subset DEG lists (= raw Seurat -"FindMarkers" -MAST outputs) for each major cell cluster, for various group comparisons (detailed in the table of contents within the file).Details on the performed calculations and nomenclature are given in the Methods and Results sections of the main text.
File Name: Supplementary Data 4 Description: Results of differentially expressed gene calculations, for oligodendrocyte lineage sub clusters.This data file contains DEGs derived from the comparisons of oligodendrocyte lineage subclusters OPC_1 vs OPC_0 and MOLIGO_1 vs MOLIGO_2, as well as all DEG counts and non-subset DEG lists (= raw Seurat -"FindMarkers" -MAST outputs) for each conserved subcluster, for various group comparisons (detailed in the table of contents within the file).Details on the performed calculations and nomenclature are given in the Methods and Results sections of the main text.
Description: Functional annotation of infarction restricted oligodendrocyte subclusters.EnrichR output tables were generated from inputting DEGs (Bonferrioni adjusted p values <0.05 & log2FC >0.6, or log2FC < -0.6) derived from OPC_1 vs. OPC_0 and MOLIGO_1 vs. MOLIGO_2 comparisons.EnrichR outputs generated from upregulated DEGs are termed UP_OPC_1 and UP_MOLIGO_1 and outputs generated from downregulated DEGs are termed DOWN_OPC_1 and DOWN_MOLIGO_1, respectively, followed by the queried database (e.g.GO_Biological_Process_2023).Binary gene by term matrices show which UP or DOWN regulated DEGs are associated to which enriched terms.Only terms with Adjusted.P.value < 0.05 were included in the binary matrices.Up to Top 20 enriched terms, sorted by descending EnrichR Combined.Scores were included in the binary matrices.
File Name: Supplementary Data 6 Description: Monocle3 derived gene modules for oligodendrocyte subclusters.This data file contains all Monocle3 derived gene modules derived from genes which robustly change as a function of pseudotime (q-values < 0.05 and morans I >0.05).Further details on the performed calculations are given in the Methods section of the main text.
File Name: Supplementary Data 7

Description:
Summary of cell count comparisons derived from immunofluorescences stainings of rat brain tissue.This data file contains the results of all between group comparisons of immunopositive cell counts within perilesional grey matter (GM), affected white matter (WM) and ischemic core regions of interest (ROI), in rat coronal brain sections.Cell counts were derived from immunofluorescence stainings, colocalizing various targets of interest, as detailed within the file.For all comparisons Kruskal-Wallis-H-Tests, followed by Dunn's post hoc comparisons were performed.
File Name: Supplementary Data 8

Description:
Summary of cell count comparisons derived from immunofluorescences stainings of mouse brain tissue.This data file contains the results of all between group comparisons of immunopositve cell counts within grey matter (GM) white matter (WM) and lesion core (core) regions of interest (ROI), in murine coronal brain sections.Cell counts were derived from immunofluorescence stainings, colocalizing various targets of interest, as detailed within the file.For all comparisons Mann-Whitney U tests were performed.