Selective isolation of mouse glial nuclei optimized for reliable downstream omics analyses

Background Isolation of cell types of interest from the brain for molecular applications presents several challenges, including cellular damage during tissue dissociation or enrichment procedures, and low cell number in the tissue in some cases. Techniques have been developed to enrich distinct cell populations using immunopanning or fluorescence activated cell/nuclei sorting. However, these techniques often involve fixation, immunolabeling and DNA staining steps, which could potentially influence downstream omics applications. New Method Taking advantage of readily available genetically modified mice with fluorescent-tagged nuclei, we describe a technique for the purification of cell-type specific brain nuclei, optimized to decrease sample preparation time and to limit potential artefacts for downstream omics applications. We demonstrate the applicability of this approach for the purification of glial cell nuclei and show that the resulting cell-type specific nuclei obtained can be used effectively for omics applications, including ATAC-seq and RNA-seq. Results We demonstrate excellent enrichment of fluorescently-tagged glial nuclei, yielding high quality RNA and chromatin. We identify several critical steps during nuclei isolation that help limit nuclei rupture and clumping, including quick homogenization, dilution before filtration and loosening of the pellet before resuspension, thus improving yield. Sorting of fluorescent nuclei can be achieved without fixation, antibody labelling, or DAPI staining, reducing potential artifactual results in RNA-seq and ATAC-seq analyses. We show that reproducible glial cell type-specific profiles can be obtained in transcriptomic and chromatin accessibility assays using this rapid protocol. Comparison with existing methods Our method allows for rapid enrichment of glial nuclei populations from the mouse brain with minimal processing steps, while still providing high quality RNA and chromatin required for reliable omics analyses. Conclusions We provide a reproducible method to obtain nucleic material from glial cells in the mouse brain with a quick and limited sample preparation. Highlights ### Competing Interest Statement The authors have declared no competing interest. * AST : astrocyte BSC : back scatter DAPI : 4′,6-diamidino-2-phenylindole DEPC : diethyl pyrocarbonate DNA : deoxyribonucleic acid DTT : dithiothreitol FACS : fluorescence-activated cell sorting FANS : fluorescence-activated nuclei sorting FSC : forward Scatter GFP : green fluorescent protein MACS : magnetic-activated cell sorting MG : microglia OLs : oligodendrocytes OPCs : oligodendrocyte precursor cells PBS : phosphate buffered saline RIN : RNA integrity number RNA : ribonucleic acid