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bioRxiv · 10.1101/2025.08.11.669607

PU.1-driven enrichment enables microglia profiling from frozen brain tissue using the high-throughput Smart-seq3xpress method

Dostalova, D.·Abaffy, P.·Rohlova, E.·Kriska, J.·Knotek, T.·Tureckova, J.·Kirdajova, D.·Anderova, M.·Valihrach, L.
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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 protocols for their specific enrichment. We introduce a method for the selective isolation of microglial nuclei from fresh-frozen brain tissue using the transcription factor PU.1 as a nuclear marker. To stabilize PU.1 for reliable detection, a brief formaldehyde fixation step is applied. The protocol is fully compatible with Smart-seq3xpress, a high-sensitivity, full-length transcriptomic method offering isoform- and allele-level resolution, making the workflow scalable and cost-efficient. 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 addresses key limitations of single-nucleus RNA - sequencing and opens new possibilities for studying microglial states in cryopreserved and archival brain tissue, broadening access to cellular insights in both basic and translational research.

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Dostalova, D., Abaffy, P., Rohlova, E., Kriska, J., Knotek, T., Tureckova, J., Kirdajova, D., Anderova, M., Valihrach, L.. 2025-08-15. PU.1-driven enrichment enables microglia profiling from frozen brain tissue using the high-throughput Smart-seq3xpress method. https://doi.org/10.1101/2025.08.11.669607

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