Isolation of nuclei from multiple brain cell types for omics interrogation

Our group is interested in understanding how environmental and cell-autonomous factors drive macrophage function throughout development and adulthood. Here, we present a nuclei isolation protocol for the epigenomic interrogation of multiple cell type populations in the human and rodent brain.

The mission of our lab is to decipher the gene regulatory mechanisms and environmental factors which orchestrate macrophage identity in health and disease states. Macrophages are highly dynamic tissue-resident innate immune cells whose primary function is to survey the surrounding environment and respond to a plethora of external stimuli. Microglia, the tissue-resident macrophages of the brain, constantly interact with neighboring cells and the extracellular matrix during both development and adulthood. Upon integrating signals from the brain environment, microglial activity encompasses a diverse set of functions, such as driving tissue remodeling and wound healing, participating as modulators of neuronal activity, and damaging neighboring cells through excessive secretion of inflammatory mediators.

We previously identified core features of human microglia transcriptomes and epigenetic landscapes (Gosselin et al., Science 2017). Microglia were isolated by gentle dissociated of fresh brain tissue from surgical resections, which enabled us to describe a microglial environment-sensitive gene expression program. However, this approach has certain limitations. Various bottlenecks include the limited availability of fresh human brain tissue, differences across brain regions, alterations in cells due to disease conditions, and difficulties isolating multiple types of live brain cells simultaneously.

To overcome these limitations, we adjusted and modified nuclei isolation protocols and developed a fluorescence activated nuclear sorting strategy to obtain distinct brain cell type populations with high purity. These isolated cell type populations can then be further studied to identify chromatin features and transcription factor binding sites (ChIP-seq), open chromatin regions (ATAC-seq), and chromatin conformation (PLAC-seq, HiChIP). By applying our protocol, we were able to decipher cell-type-specific promoter-enhancer interactomes which allowed for the interpretation of genetic risk alleles and variants that are associated with brain disorders like Alzheimer’s disease (Nott et al., Science 2019).

Here, we lay out in detail our protocol to isolate nuclei of multiple cell types from the human and mouse brain. Our nuclei isolation protocol is compatible with postmortem tissue, which enables translational examination of archived brain samples, in particular samples of certain disease pathologies. Our protocol can be expanded to isolate nuclei from different species and non-brain cell types (Nott et al., Nature Protocols 2020).  For example, we isolated peripheral PU.1-positive myeloid cells from a Schwannoma tumor and mapped the enhancer landscape of this distinct population.

Collectively, our protocol can be applied with ease as a method to answer questions regarding histone modifications, transcription factor binding, chromatin accessibility and chromosome architecture in health and disease. We hope that others will find this a useful, accessible tool for answering questions about specific cell populations both in homeostasis and disease.