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Updated: Jun 21, 2025
Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing
Published on: December 3, 2019
In situ Patch-seq analysis of microglia reveals a lack of stress genes as found in FACS-isolated microglia
Olga Bakina1,2, Thomas Conrad3, Nina Mitic4
1Cellular Neurosciences, Max-Delbrück Center for Molecular Medicine, Berlin, Germany.
Abstract:
We applied the patch-seq technique to harvest transcripts from individual microglial cells from cortex, hippocampus and corpus callosum of acute brain slices from adult mice. After recording membrane currents with the patch-clamp technique, the cytoplasm was collected via the pipette and underwent adapted SMART-seq2 preparation with subsequent sequencing. On average, 4138 genes were detected in 113 cells from hippocampus, corpus callosum and cortex, including microglia markers such as Tmem119, P2ry12 and Siglec-H. Comparing our dataset to previously published single cell mRNA sequencing data from FACS-isolated microglia indicated that two clusters of cells were absent in our patch-seq dataset. Pathway analysis of marker genes in FACS-specific clusters revealed association with microglial activation and stress response. This indicates that under normal conditions microglia in situ lack transcripts associated with a stress-response, and that the microglia-isolation procedure by mechanical dissociation and FACS triggers the expression of genes related to activation and stress.
Insights
Patch-seq reveals that healthy mouse microglia in brain slices lack stress-response genes. Microglia isolation using FACS triggers stress and activation gene expression, suggesting current methods impact cell state.
Area of Science:
- Neuroscience
- Cell Biology
- Genomics
Background:
- Microglia are crucial immune cells in the central nervous system.
- Understanding microglia gene expression in their native environment is essential.
- Previous studies often relied on methods that may alter microglial state.
Purpose of the Study:
- To profile the transcriptomes of individual microglia in situ using patch-seq.
- To compare gene expression in microglia within acute brain slices versus isolated cells.
- To identify potential artifacts introduced by cell isolation procedures.
Main Methods:
- Patch-seq was used to harvest transcripts from single microglial cells in mouse brain slices (cortex, hippocampus, corpus callosum).
- Patch-clamp electrophysiology was performed before cytoplasm collection.
- Adapted SMART-seq2 protocol was used for library preparation and subsequent sequencing.
Main Results:
- An average of 4138 genes were detected per cell across 113 analyzed microglia.
- Standard microglia markers (Tmem119, P2ry12, Siglec-H) were identified.
- Two cell clusters, absent in the patch-seq data, were found in previously published FACS-isolated microglia datasets.
- Pathway analysis linked these absent clusters to microglial activation and stress responses.
Conclusions:
- In situ microglia in healthy adult mice do not express transcripts associated with stress responses.
- Mechanical dissociation and Fluorescence-Activated Cell Sorting (FACS) for microglia isolation induce activation and stress gene expression.
- Patch-seq offers a valuable method to study microglia in their native physiological state, minimizing isolation-induced artifacts.
