Engineering Microfluidic Systems for Low-Input Multi-Omic Profiling of Brain Regulatory States

Abstract

Microfluidic systems enable sensitive application of sequencing based "omic" assays to rare sample sets. In this thesis, we developed and applied these systems to address fundamental limitations in existing assays and to investigate biological questions that are inaccessible using traditional bulk protocols. First, we introduced a microfluidic platform for rapid field-ready library preparation of viral samples for nanopore sequencing. Our microreactor enabled laboratory independent viral diagnostics of field-collected Senecavalley virus A samples with accuracy in-line with gold standard RT-PCR assays. Additionally, our system enabled real-time mutation identification, detecting two high-confidence consensus single nucleotide variants within the SVA positive cohort. We then applied microfluidic MOWChIP platform to two epigenomic investigations into the effects of psilocybin on mouse cortical and subcortical regions. First, we examined the sex-specific enhancer alterations following psilocybin exposure in neurons extracted from the frontal cortex and nucleus accumbens to elucidate therapeutic mechanisms for combating opioid use disorder. Here we describe localized increases in enhancer activity in the mesolimbic nucleus accumbens compared to the frontal cortex. Additionally, we demonstrate significant recovery of key enhancer linked gene pathways disrupted by prolonged opioid use following acute psilocybin exposure in male mice absent in female cohort. Finally, we investigated epigenetic inheritance of prolonged psilocybin exposure in prenatal dams on their offspring. We uncover significant transcription factor regulatory network disruptions that persist through to the F1 generation. Specifically, reduction in regulatory efficacy of Egr2 and JunB transcription factors of which direct psilocybin exposure mediates significant expressional increases in the prefrontal cortex. We further uncover a sex specific nature of these alterations, in which minimized reach of early gene Egr2 is found primarily in female offspring following maternal exposure. These multi-omic investigations uncover significant sex-specific implications of psilocybin exposure on brain epigenome.