Abstract
<p>Increased understanding of the functional complexity of the genome has led to growing recognition about the role of epigenetic/transcriptional variation in health and disease. Current analyses of the human brain, however, are limited by the use of “bulk” tissue, comprising a heterogeneous mix of different neural cell types. Because epigenetic processes play a critical role in determining cell type-specific patterns of gene regulation it is important to consider cellular composition in regulatory genomic studies of human post-mortem tissue, and there is a need for methods to purify populations of specific cell-types. This protocol builds on a previous protocol that uses fluorescence-activated nuclei sorting (FANS) to isolate and profile nuclei from multiple different human brain cell-types from frozen post-mortem tissue. Because NeuN is not an optimal marker for neuronal nuclei from fetal cortex, we have optimized a method using a SATB2 antibody to purify nuclei from excitatory neurons in both fetal and postnatal cortex. Purified populations of nuclei are amenable to simultaneous profiling of i) DNA modifications (via bisulfite sequencing / array), ii) histone modifications (via CUT&Tag), iii) open chromatin analysis (via ATAC-seq), and iv) gene expression (via RNA-seq).</p>