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

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.