Brain cell type nuclei enrichment without fixative for nanoCUT&Tag and other omics approaches

Abstract We present a workflow for cell-type-enriched epigenomic profiling of the neurovascular unit and associated cell types, including brain endothelial cells, mural cells, microglia, astrocytes, neurons, and oligodendrocytes isolated from frozen unfixed human and mouse brain tissue. The workflow consists of an unfixed nuclei isolation with improved vascular nuclei release, fluorescence-activated nuclei sorting (FANS) based on cell-type-specific DNA-bound proteins, and nanoCUT&Tag for epigenomic profiling. The nanoCUT&Tag uses Tn5 transposase fused to a single-chain nanobody with secondary antibody-like properties. This allows low-input epigenomic profiling that is compatible with FANS-enriched nuclei immunolabeled for transcription factor markers, which is not possible with traditional CUT&Tag approaches. The protocol allows studying the human brain epigenome in a cell type-specific manner, which is increasingly associated with neurodegenerative diseases. The workflow can be used on various tissue sources, including resected and post-mortem archived brain tissue and can be coupled to multiple-omics approaches including single nuclei (sn)RNA-seq, snATAC-seq, and proteomics.