Isolation and Molecular Profiling of Nuclei of Specific Neuronal Types from Human Cerebral Cortex and Striatum

Most pathological conditions of the central nervous system do not affect all cell types to the same extent. Delineation of molecular events underlying disease symptoms, including genetic, epigenetic, and transcriptional changes, thus relies on the ability to characterize a specific cell type separately from others. We have developed a methodology for the collection of nuclear RNA and genomic DNA of specific cell types from frozen post-mortem striatum and cerebral cortex. This allows deep transcriptomic profiling of specific cell populations and characterization of their genomes and epigenetic properties. The method is based on the purification of cell nuclei, followed by fluorescence-activated sorting of nuclei (FANS) labeled with nucleic acid probes or antibodies binding to targets present in specific cell types. The protocol describes in detail the procedure for isolating and labeling neuronal and glial nuclei from human brain tissue, the steps that can be taken to extract RNA and genomic DNA, a way to combine the procedure with ATAC-seq to yield information about chromatin accessibility, as well as computational measures for assessing the quality of cell type-specific RNA-seq and ATAC-seq datasets. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tissue homogenization, isolation of cell nuclei by ultracentrifugation and formaldehyde-fixation Basic Protocol 2: Antibody-based labeling and isolation of nuclei of specific neocortical neuron types Support Protocol 1: Generation of ATAC-seq libraries from the nuclei of specific neuron types of the cerebral cortex Basic Protocol 3: Nucleic acid hybridization-based labeling and isolation of nuclei of specific striatal projection neuron types Alternate Protocol 1: Labeling and isolation of nuclei of specific striatal interneuron types Support Protocol 2: Generation of ATAC-seq libraries from the nuclei of specific striatal neuron types Basic Protocol 4: Extraction of genomic DNA and nuclear RNA and preparation of sequencing libraries Basic Protocol 5: Processing and quality control of FANS-seq and ATAC-seq data.