Cryopreservation of microglia enables single-cell RNA sequencing with minimal effects on disease-related gene expression patterns
Brenda Morsey,
Meng Niu,
Shetty Ravi Dyavar,
Courtney V. Fletcher,
Benjamin G. Lamberty,
Katy Emanuel,
Anna Fangmeier,
Howard S. Fox
Affiliations
Brenda Morsey
Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA
Meng Niu
Department of Genetics, Cell Biology and Anatomy; and Bioinformatics and Systems Biology Core, University of Nebraska Medical Center, Omaha, NE 68198, USA
Shetty Ravi Dyavar
Antiviral Pharmacology Laboratory, UNMC Center for Drug Discovery, University of Nebraska Medical Center, Omaha, NE 68198, USA
Courtney V. Fletcher
Antiviral Pharmacology Laboratory, UNMC Center for Drug Discovery, University of Nebraska Medical Center, Omaha, NE 68198, USA
Benjamin G. Lamberty
Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA
Katy Emanuel
Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA
Anna Fangmeier
Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA
Howard S. Fox
Department of Neurological Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA; Corresponding author
Summary: Microglia play a key role in brain development, normal homeostasis, and neurodegenerative disorders. Single-cell technologies have led to important findings about microglia, with many animal model studies using single-cell RNA sequencing (scRNA-seq), whereas most human specimen studies using archived frozen brains for single-nucleus RNA sequencing (snRNA-seq). However, microglia compose a small proportion of the total brain tissue; snRNAseq depletes expression of microglia activation genes that characterize many diseases. Here we examine the use of purified, cryopreserved microglia for scRNA-seq. Comparison of scRNA-seq on paired fresh and cryopreserved microglia from rhesus monkeys revealed a high level of correlation of gene expression between the two conditions. Disease-related genes were relatively unaffected, but an increase in immediate-early gene expression was present in cryopreserved cells. Regardless, changes in immediate-early gene expression are still detectable. Cryopreservation of microglia is a suitable procedure for prospectively archiving samples.
