Knotek, T. | bioRxiv Science

bioRxiv2025

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

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.

neuroscience↗

bioRxiv2023

Astrocyte-like subpopulation of NG2 glia in the adult mouse cortex exhibits characteristics of neural progenitor cells and is capable of forming neuron-like cells after ischemic injury

Glia cells expressing neuron-glial antigen 2 (NG2) play a critical role as oligodendrocyte precursor cells (OPCs) in the healthy brain; however, their differentiation potential after ischemic injury remains an unresolved question. Here, we aimed to elucidate the heterogeneity and role of NG2 glia in the ischemic brain. We used transgenic mice to label NG2-expressing cells and their progeny with red fluorescent protein tdTomato in the healthy brains and those after focal cerebral ischemia (FCI). Based on single-cell RNA sequencing, the labeled glial cells were divided into five distinct subpopulations. The identity of these subpopulations was determined based on gene expression patterns. In addition, membrane properties were further analyzed using the patch-clamp technique. Three of the observed subpopulations represented OPCs, whereas the fourth group exhibited characteristics of cells destined for oligodendrocyte fate. The fifth subpopulation of NG2 glia carried astrocytic markers. Importantly, we detected features of neural progenitors in these cells. This subpopulation was present in both healthy and post-ischemic tissue; however, its gene expression changed after ischemia, with genes related to neurogenesis being more abundant. Neurogenic gene expression was monitored over time and complemented by immunohistochemical staining, which showed increased numbers of Purkinje cell protein 4-positive NG2 cells at the edge of the ischemic lesion 12 days after FCI, and NeuN-positive NG2 cells 28 days after injury, indicating the existence of neuron-like cells that develop from NG2 glia in the ischemic tissue. Our results provide further insight into the differentiation plasticity and neurogenic potential of NG2 glia after stroke. Main PointsO_LIDifferent subpopulations of NG2 glia in the healthy and ischemic adult cortex were identified based on their gene expression and membrane properties. C_LIO_LIAstrocyte-like NG2 glia exhibit neurogenic gene expression and are more abundant in post-ischemic tissue. C_LIO_LIProgeny of NG2-positive cells carrying neuronal marker NeuN was observed at the edge of the ischemic lesion. C_LI

neuroscience↗