Abstract: Objective: Monoamine system plays a key role in the pathophysiology of a wide range of neuropsychiatric disorders including Parkinson’s, Huntington’s, drug addiction and many other conditions. Of note, there is a large amount of literatures exploring monoamine pathways in the context of adaptive mechanisms inside the cell nucleus, including changes in gene expression and alterations in chromatin modifications, structure and functions. However, such types of studies were largely focused on forebrain structures that receive input from monoaminergic neurons. Comprehensive genome-scale and cell-type specific mappings is lacking for brainstem areas that consist of monoaminergic neurons, including the substantia nigra (SN) and ventral tegmental area (VTA). This is especially the case for human study, due to the limited access to clinical human brain samples and lack of efficient assay for cell-type specific isolation from postmortem brain tissues after low-temperature or fixative preservations. Here, we introduce novel protocol to comprehensively map the 3D genome and nuclear transcriptome in selective population of dopaminergic neurons isolated from human midbrain. Methods: Mammalian chromatin is highly packed and compartmentalized into Topologically Associated Domains (TADs). Although TAD structures were reported to conserve in between species and cell types, chromatin contacts within TAD were often highly specific for different cell types in brain, and were implicated in the genetic risk of psychiatric disorders. Fragmentation-religation based DNA-DNA proximity (Hi-C) assays were designed to detect chromosomal conformations, which provide an additional layer of epigenomic regulation on top of previous well-studied epigenetic mechanisms including DNA and histone modifications. However, current Hi-C protocols either require millions of cells as input, or only offer limited resolution when applied to low input material, particularly when probing rare cell types from human postmortem brain tissue. Here, we designed Tn5-Hi-C, a modified Hi-C protocol that involves Transposase-based chromatin fragmentation, bypass the low-efficient and costly steps of biotin pull-down and adaptor-based library preparation, and was applicable to as low as 5000 nuclei as input. We performed Tn5-Hi-C on dopaminergic chromatin subsampling by fluorescence-activated sorting from human substantia nigra, delivered chromosomal contact maps at resolution similar to conventional Hi-C, while decreasing the input by three orders of magnitude.  Results: For the first time, we provided genome-wide mapping of 3D genome (Tn5-Hi-C) and transcriptome (nuclear RNA-seq) profiles in human dopaminergic neurons from substanina nigra, identified hundreds of chromosomal contacts associated with higher-order open chromatin and differential gene expression, including genes from regulatory networks critical for dopaminergic differentiation, maintenance and protection from neurodegeneration. Conclusion: These findings will provide unique features of dopaminergic neuronal epigenome and could illuminate the role of higher order chromatin in transcriptional regulation in adult human midbrain. The capacity of the protocols presented here could easily be scaled up to probe large disease cohorts, offering novel insights into the genomic organization and disease-associated alterations in brainstem monoamine neurons along with other rare brain cell populations assigned with critical roles in the regulation of human cognition and behavior.

Key words: chromosome conformation, Hi-C, Tn5 transposase, dopaminergic neuron, substantia nigra