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<p>Single-cell transcriptomics has revealed the central role of microglia in brain development, homeostasis, and disease, particularly in the context of neuroinflammation. While single-cell RNA-sequencing enables targeted microglial analysis from fresh tissue, studying these cells in cryopreserved or archival samples remains challenging due to the lack of a protocol for their specific enrichment and RNA-sequencing. In this study, we developed a method for profiling microglial nuclei from fresh-frozen tissue using Smart-seq3xpress. This approach relies on PU.1-based enrichment, made possible by a brief formaldehyde fixation step to preserve the antigen. To ensure compatibility with Smart-seq3xpress, we utilized a Thermolabile Proteinase K treatment to reverse cross-links, thereby maintaining high transcriptomic sensitivity. We benchmarked the method in a mouse model of ischemic stroke, evaluating both technical performance and its ability to capture biologically meaningful microglial states. Compared to standard single-nucleus protocols, our approach yielded higher gene and UMI counts and a greater proportion of coding reads. Transcriptomic profiles closely matched those from whole-cell RNA-sequencing, including the detection of activation markers and diverse microglial subpopulations. This approach enables high-resolution transcriptomic analysis of microglia from fresh-frozen or archival brain samples, overcoming major limitations of single-nucleus RNA sequencing. It broadens access to cellular insights from biobanked material, supporting both basic research and translational studies of neuroinflammatory disease.</p> Graphical Abstract <p></p> <p>A modified Smart-seq3xpress protocol was developed for microglial profiling by combining formaldehyde fixation and PU.1-based nuclei enrichment from fresh-frozen brain tissue. The method yields high gene and UMI counts alongside strong coding coverage, enabling high-resolution characterization of diverse microglial activation states following stroke.</p>

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Adapted Smart-seq3xpress Facilitates Selective Microglial Transcriptomic Profiling From Frozen Brain Tissue

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年5月14日

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  • Dominika Dostalova,
  • Pavel Abaffy,
  • Eva Rohlova,
  • Jan Kriska,
  • Tomas Knotek,
  • Jana Tureckova,
  • Denisa Kirdajova,
  • Miroslava Anderova,
  • Lukas Valihrach

摘要

Single-cell transcriptomics has revealed the central role of microglia in brain development, homeostasis, and disease, particularly in the context of neuroinflammation. While single-cell RNA-sequencing enables targeted microglial analysis from fresh tissue, studying these cells in cryopreserved or archival samples remains challenging due to the lack of a protocol for their specific enrichment and RNA-sequencing. In this study, we developed a method for profiling microglial nuclei from fresh-frozen tissue using Smart-seq3xpress. This approach relies on PU.1-based enrichment, made possible by a brief formaldehyde fixation step to preserve the antigen. To ensure compatibility with Smart-seq3xpress, we utilized a Thermolabile Proteinase K treatment to reverse cross-links, thereby maintaining high transcriptomic sensitivity. We benchmarked the method in a mouse model of ischemic stroke, evaluating both technical performance and its ability to capture biologically meaningful microglial states. Compared to standard single-nucleus protocols, our approach yielded higher gene and UMI counts and a greater proportion of coding reads. Transcriptomic profiles closely matched those from whole-cell RNA-sequencing, including the detection of activation markers and diverse microglial subpopulations. This approach enables high-resolution transcriptomic analysis of microglia from fresh-frozen or archival brain samples, overcoming major limitations of single-nucleus RNA sequencing. It broadens access to cellular insights from biobanked material, supporting both basic research and translational studies of neuroinflammatory disease.

Graphical Abstract

A modified Smart-seq3xpress protocol was developed for microglial profiling by combining formaldehyde fixation and PU.1-based nuclei enrichment from fresh-frozen brain tissue. The method yields high gene and UMI counts alongside strong coding coverage, enabling high-resolution characterization of diverse microglial activation states following stroke.