Enzymatic dissociation induces transcriptional and proteotype bias in brain cell populations

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 artefacts when implementing enzymatic digestion-based isolation methods for brain cell analyses. Overall design: Adult male mice hippocampi where dissociated via either enzymatic digestion for 30 min at 37°C or via mechanical dissociation at 4°C. The resulting cell suspensions were analysed via 10X Genomics single cell sequencing.