Abstract
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 °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 °C. These findings emphasize the risk of introducing technical biases and biological artifacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.
Highlights
The neuroscience field is continuously implementing novel techniques for gene sequencing, epigenetic analyses, and proteotype profiling at a rapidly increasing rate
Our study aimed at systematically investigating the possible influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue, thereby taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia
We found that 30 min ED37◦ was associated with altered expression of genes related to cellular metabolic processes and energy expenditure pathways in both microglia and astrocytes
Summary
The neuroscience field is continuously implementing novel techniques for gene sequencing, epigenetic analyses, and proteotype profiling at a rapidly increasing rate. The main techniques available to obtain single-cell suspensions from brain tissue are enzymatic digestion (ED) and mechanical dissociation (MD) The latter is typically carried out at 4 ◦C [1], whereas ED requires incubation at temperatures between 30–37 ◦C [2,3,4]. Unlike MD at cold temperatures, which largely spares the cellular transcriptome and proteome [7], the ED technique may introduce additional biological biases to the transcriptional and proteotype profiles of cells. The latter technique might influence the relative amount of cell surface markers, as changes in surface receptor trafficking might occur during enzymatic digestions at a temperature where cells are metabolically active. This, in turn, may introduce additional bias in cell sorting analyses such as fluorescence-activated cell sorting (FACS) and cytometry by time of flight (CyTOF)
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