For All the Primate FANS: Optimized Isolation of Nuclei from Frozen Postmortem Primate Brain for Fluorescence-Assisted Nuclei Sorting (FANS)

Authors

Elaine E. Guevara, Department of Evolutionary Anthropology, Duke University, Durham, NC, USA.
Begün Erbaba, Department of Anthropology and Center for the Advanced Study of Human Paleobiology, The George Washington University, Washington DC, USA.
Gregory M. Cresswell, School of Medicine and Health Sciences, The George Washington University, Washington DC, USA.
Melissa K. Edler, Department of Anthropology, School of Biomedical Sciences, and Brain Health Research Institute, Kent State University, Kent, OH, USA.
Chet C. Sherwood, Department of Anthropology and Center for the Advanced Study of Human Paleobiology, The George Washington University, Washington DC, USA.

Document Type

Journal Article

Publication Date

6-1-2025

Journal

The Yale journal of biology and medicine

Volume

98

Issue

2

DOI

10.59249/LLUJ2834

Keywords

chimpanzee; cortex; flow sorting; methylation; nonhuman primate; single cell genomics

Abstract

Epigenetic alterations are cell type-specific and require methods like single cell sequencing and cell type sorting by flow cytometry. These methods often rely on the availability of fresh tissue, yet postmortem frozen tissue is typically the only material available from non-experimental subjects, including humans and other nonhuman primates (NHP). Many insights can be gained from analysis of these precious samples. To this end, we developed a protocol for isolating intact nuclei from small starting amounts of postmortem frozen chimpanzee (Pan troglodytes) cerebral cortex tissue. Isolated nuclei can be input directly into single cell epigenomics protocols like ATAC-seq or can be immunostained for enrichment of neuronal nuclei via fluorescent-activated nuclei sorting (FANS) followed by bulk epigenetic methods like methylome sequencing. We adapted and optimized this protocol based on existing human brain tissue protocols. Our protocol specifically addresses challenges presented by postmortem frozen NHP brain tissue, including high levels of myelin debris and reduced RNA integrity. We include key steps and troubleshooting guidance to improve nuclei quality and sorting outcomes, and we also discuss limitations and considerations for researchers interested in using these methods.

APA Citation

Guevara, Elaine E.; Erbaba, Begün; Cresswell, Gregory M.; Edler, Melissa K.; and Sherwood, Chet C., "For All the Primate FANS: Optimized Isolation of Nuclei from Frozen Postmortem Primate Brain for Fluorescence-Assisted Nuclei Sorting (FANS)" (2025). GW Authored Works. Paper 7350.
https://hsrc.himmelfarb.gwu.edu/gwhpubs/7350

Department

Medicine