GSE162663
Cryopreservation of microglia enables single-cell RNA sequencing with minimal effects on disease-related gene expression patterns
We report the effect of cryopreservation on isolated microglia and macrophages from the brains of rhesus monkeys. At the time of necropsy, cells were isolated from the brain and either used fresh or cryopreserved. For single cell sequencing, isolates were purified for CD11b expression and captured and processed through the 10xGenomics platform following by sequencing. We find that cryopreservation altered the expression of approximately 2% of the genes. These were not enriched for any known microglia/macrophage set of differentially regulated genes, but was enriched in immediate early genes and the acute phase pathway. Assessment of the fresh and cryopreseved cells in comparison to a prior dataset from animals with encephalitis revelaed equal ability to detect differentially expresed genes. Thus, given the practical difficulty of freshly processing microglia from human and non-human primate brains for isolation and scRNAseq processing the same day, and the ability to archive sampoles for potential use in scRNAseq experiments, cryopreservation is an optimal choice for future analysis of these important brainn cells, and likely other cell types. Overall design: Rhesus macaques were infected with SIVmac251.  Five weeks post infection, one animal was treated with a two-drug cART consisting of 8mg/ml dolutegravier (DTG), 80 mg/ml emtricitabine (FTC) or a four-drug cART, consisting of 4 mg/ml DTG, 1mg/ml tenofovir alafenamide fumerate (TAF), 40 mg/ml FTC, 12 mg/ml maraviroc (MVC). Animals remained on drug regime for 6 months post latency (<50 SIV copies/ml plasma) and were sacrificed as per experimental design. MIcroglia were isolated and either further purified and subjected to capture and library production, or cryopreserved followed by thawing, purification, capute and library preparation. LIbraries were then subjected to sequencing.