Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 21, Issue 21, Pages -Publisher
MDPI
DOI: 10.3390/ijms21217944
Keywords
microglia; astrocytes; neurons; enzymatic digestion; single-cell sequencing; protocol; microglia isolation
Funding
- Swiss National Science Foundation [310030_188524, PZ00P3_180099, 31003A_160259]
- Forschungskredit from the University of Zurich [K-51503-05-01]
- European Research Council (ERC StGrant) [804949]
- Synapsis Foundation
- ETH [ETH-25 15-2]
- Novartis Foundation for Medical-Biological Research
- Personalized Health and Related Technologies (PHRT) strategic focus area of ETH
- Novartis Foundation for Medical-Biological Research [16C231]
- Swiss Cancer Research [KFS-3852-02-2016, KFS-4146-02-2017]
- Swiss National Science Foundation (SNF) [PZ00P3_180099] Funding Source: Swiss National Science Foundation (SNF)
- European Research Council (ERC) [804949] Funding Source: European Research Council (ERC)
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Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here, we provide a systematic investigation of the influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia. We show that standard enzymatic digestion of brain tissue at 37 degrees C induces profound and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared to an optimized mechanical dissociation protocol at 4 degrees C. These findings emphasize the risk of introducing technical biases and biological artifacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.
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