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Minimizing the ex vivo confounds of cell-isolation techniques on transcriptomic profiles of purified microglia

ABSTRACT: Minimizing the ex vivo confounds of cell-isolation techniques on transcriptomic profiles of purified microglia

PROVIDER: PRJNA744767 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR15069400_1.fastq.gz	Fastqsanger.gz
	SRR15069400_2.fastq.gz	Fastqsanger.gz
	SRR15069401_1.fastq.gz	Fastqsanger.gz
	SRR15069401_2.fastq.gz	Fastqsanger.gz
	SRR15069402_1.fastq.gz	Fastqsanger.gz

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Minimizing the ex vivo confounds of cell-isolation techniques on transcriptomic profiles of purified microglia

Project description:Modern molecular neuroscience studies require analysis of specific cellular populations derived from brain tissue samples to disambiguate cell-type specific events. This is particularly true in glial cell types, such as microglia, which as minority cell populations in the brain, may be obscured in whole tissue analyses. Microglia have central functions in development, aging, and neurodegeneration and are a current focus of neuroscience research. A long-standing concern for glial biologists using in vivo models is whether cell isolation from CNS tissue could introduce ex vivo artifacts in microglia, which respond quickly to changes in the environment. Mouse microglia were purified by antibody-conjugated magnetic bead, and cytometer- and cartridge-based fluorescence-activated cell sorting approaches to compare and contrast performance. The Cx3cr1-NuTRAP (nuclear tagging and translating ribosome affinity purification) model was used to provide an endogenous fluorescent microglial marker and a microglial-specific translatome profile lacking cell isolation artifacts. All methods performed similarly for microgial purity with main differences being in cell yield and time of isolation. Ex vivo activation signatures occurred principally during the initial tissue dissociation and cell preparation and not the microglial cell sorting. Utilizing transcriptional and translational inhibitors during the cell preparation prevented the activational phenotype. These data demonstrate that a variety of microglial isolation approaches can be used, depending on experimental needs, and that inhibitor cocktails are effective at reducing cell preparation artifacts.

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