ArticleCellular and molecular neurobiology2026
Adapted Smart-seq3xpress Facilitates Selective Microglial Transcriptomic Profiling From Frozen Brain Tissue.
Article in Cellular and molecular neurobiology, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
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.
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