Isolation of Neuronal Nuclei From Human Frozen Post-Mortem Prefrontal Cortex Brain Tissue Using Fluorescence-Activated Nuclei Sorting (FANS) For Methylation Analyses v1

Abstract

There is a need for disentangling the role of transcriptional and epigenetic variation in mental health, disease, and mortality. Although alterations in DNA methylation patterns have been found to be particularly stable and highly concentrated in the brain, the study of bulk tissue, which contains several types of cells, can be limited and potentially significantly mask different alterations driven by specific cell types. Therefore, there is a need for methods to be able to purify specific cell-type populations that can be used in downstream molecular analyses. This protocol describes a methodology that uses fluorescence-activated nuclei sorting (FANS) to overcome these limitations and analyze, specifically, neuronal nuclei from frozen post-mortem prefrontal cortex tissue. Specifically, this protocol was based on recently-published protocols with the same goal with adaptations that have proven to improve yield and quality of the samples in our laboratory using the BD FACSJazzTM cell sorting system. To our knowledge, this is the only published protocol where a methodology for a flow cytometry instrument with a manual laser and sorting alignment is described in detail. This method can be used to purify populations of neuronal (NeuN+) and non-neuronal (NeuN-) nuclei from adult frozen post-mortem brain tissue, with tissue samples yielding purified populations of nuclei amenable to be able to perform analyses of DNA methylation, genotyping, and potentially, open chromatin analysis (via ATAC-seq).