Microglia‑enriched single‑cell and single‑nucleus RNA‑seq dataset from a mouse model of focal cerebral ischemia induced by permanent middle cerebral artery occlusion

Dataset Description

This repository contains pre‑processed single‑cell (scRNA-seq) and single‑nucleus RNA‑seq (snRNA-seq) data supporting the publication:
“Adapted Smart-seq3xpress Facilitates Selective Microglial Transcriptomic Profiling From Frozen Brain Tissue” (DOI: 10.1007/s10571-026-01743-5).

The dataset is generated from microglial enrichment experiments using three isolation protocols: LiveCells, LiveNuclei, and FixedNuclei.

• Processing scripts are available on GitHub.
• Experimental protocols for FixedNuclei is available at Protocol.io.

Experimental Overview

Enriching microglia from fresh‑frozen brain tissue is challenging due to marker degradation and cellular fragility. To overcome these limitations, we developed a novel protocol (FixedNuclei) that selectively enriches PU.1⁺ nuclei:

• FixedNuclei – snRNA‑seq of nuclei from fresh‑frozen tissue, lightly formaldehyde-fixed, and subsequently enriched for PU.1+ nuclei; capturing nuclear transcripts only.

Results obtained with FixedNuclei protocol were compared with two commonly used protocols:

• LiveCells – scRNA‑seq of cells from papain‑dissociated fresh tissue and enriched for CD11b⁺ populations; capturing both cytoplasmic and nuclear transcripts.
• LiveNuclei – snRNA‑seq of nuclei from the CD11b⁺ fraction of LiveCells; capturing nuclear transcripts only.

Sample Collection and Library Preparation

• Samples – cortical tissue was collected from male mice subjected to permanent middle cerebral artery occlusion (7 days post‑stroke) and controls (sham).

• Library Preparation – full‑length mRNA libraries were generated using Smart‑seq3xpress (Hagemann-Jensen et al., 2022):

  • FixedNuclei – included Thermolabile Proteinase K for gentle decrosslinking while maintaining RNA integrity.

  • LiveCells / LiveNuclei – prepared using the standard Smart‑seq3xpress workflow without Thermolabile Proteinase K.

Data Processing

The pre‑processed data are provided in this repository are organized into two separate experiments:

1. main experiment (Fig. 1-4; SFig. 2, 3A,C, 4, 5).
2. repeated experiment (SFig. 3B).

For Seurat object processing, the following files are included:

• Smartseq3xpress.dgecounts.rds
• metadata.csv
• Smartseq3xpress.gene_names.txt
• Smartseq3xpress.readspercell.txt
• Smartseq3xpresskept_barcodes_binned.txt.BCUMIstats.txt
• 1DP_00_seurat_all_cells_metadata_for_subset_mg.csv (used only for generating a Seurat object with the microglia subset in 1. main experiment)

The following Seurat objects are provided (markdown files used for generating Seurat objects):

• 1. main experiment – all isolated cells  (1DP_00_all_cells_raw_data_to_seurat_merged_integrated_UMAP.Rmd).
• 1. main experiment – microglial subset  (1DP_01_mg_cells_raw_data_to_seurat_merged_integrated_UMAP.Rmd).
• 2. repeated experiment – all isolated cells (1DP_05_all_cells_LC_FN_raw_data_to_seurat_merged_integrated_UMAP.Rmd).

For library quality controls (Library_qc), the following files are included:

• LiveNuclei_raw.cram
• LiveCells_raw.cram
• FixedNuclei_raw.cram
• additional_sequence_annot.gtf

The main experiment can be explored online via the Nygen portal - all isolated cells and microglial subset.

Reference:
Hagemann-Jensen, M., Ziegenhain, C., Sandberg, R., 2022. Scalable single-cell RNA sequencing from full transcripts with Smart-seq3xpress. Nat. Biotechnol. 29, 1452–1457. https://doi.org/10.1038/s41587-022-01311-4