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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.<\/jats:p>\n                  <jats:p>\n                    <jats:bold>Graphical Abstract<\/jats:bold>\n                  <\/jats:p>\n                  <jats: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.<\/jats:p>","DOI":"10.1007\/s10571-026-01743-5","type":"journal-article","created":{"date-parts":[[2026,5,14]],"date-time":"2026-05-14T11:24:56Z","timestamp":1778757896000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Adapted Smart-seq3xpress Facilitates Selective Microglial Transcriptomic Profiling From Frozen Brain Tissue"],"prefix":"10.1007","volume":"46","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-2303-2370","authenticated-orcid":false,"given":"Dominika","family":"Dostalova","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7571-8880","authenticated-orcid":false,"given":"Pavel","family":"Abaffy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6190-4837","authenticated-orcid":false,"given":"Eva","family":"Rohlova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5684-5828","authenticated-orcid":false,"given":"Jan","family":"Kriska","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1296-155X","authenticated-orcid":false,"given":"Tomas","family":"Knotek","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1643-751X","authenticated-orcid":false,"given":"Jana","family":"Tureckova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1022-2155","authenticated-orcid":false,"given":"Denisa","family":"Kirdajova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9030-4571","authenticated-orcid":false,"given":"Miroslava","family":"Anderova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6704-4337","authenticated-orcid":false,"given":"Lukas","family":"Valihrach","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,5,14]]},"reference":[{"key":"1743_CR1","doi-asserted-by":"publisher","first-page":"451","DOI":"10.1038\/s41588-024-02036-7","volume":"57","author":"T Abay","year":"2025","unstructured":"Abay T, Stickels RR, Takizawa MT et al (2025) Transcript-specific enrichment enables profiling of rare cell states via single-cell RNA sequencing. 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