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Updated: Oct 6, 2025
Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing
Published on: December 3, 2019
Selective isolation of mouse glial nuclei optimized for reliable downstream omics analyses
Miguel A Pena-Ortiz1, Sarfraz Shafiq1, Megan E Rowland2
1Departments of Anatomy and Cell Biology, Paediatrics, and Oncology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada; Division of Genetics and Development, Children's Health Research Institute, London, ON, Canada.
This study presents a rapid method for isolating pure glial cell nuclei from mouse brains. This technique minimizes artifacts and yields high-quality RNA and chromatin for omics applications like ATAC-seq and RNA-seq.
Area of Science:
- Neuroscience
- Molecular Biology
- Genomics
Background:
- Isolating specific brain cell types for molecular studies is challenging due to potential cell damage and low yields.
- Existing methods like immunopanning or cell sorting may introduce artifacts through fixation or staining, impacting downstream omics analyses.
Purpose of the Study:
- To develop a rapid and reproducible method for purifying cell-type specific brain nuclei from genetically modified mice.
- To optimize nuclei isolation to minimize sample preparation time and artifacts for omics applications.
Main Methods:
- Utilized genetically modified mice with fluorescently-tagged nuclei for cell-type specific purification.
- Developed a streamlined nuclei isolation protocol involving quick homogenization, dilution, and specific resuspension techniques.
- Enabled nuclei sorting without fixation, antibody labeling, or DAPI staining.
Main Results:
- Achieved excellent enrichment of fluorescent glial nuclei, producing high-quality RNA and chromatin.
- Identified critical steps to prevent nuclei rupture and clumping, enhancing yield.
- Demonstrated reproducible glial cell type-specific profiles in transcriptomic and chromatin accessibility assays.
Conclusions:
- Developed a rapid, reproducible method for isolating glial nuclei from the mouse brain.
- The protocol requires minimal sample preparation and yields high-quality nucleic material suitable for omics analyses.
