Spatial Core (Moffit)

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Spatially resolved, single-cell transcriptomic techniques are an exciting and emerging toolbox for the creation of molecularly defined, cellularly resolved, and, often, functionally annotated tissue atlases. While offering the potential for transformative new insights into a wide range of tissues and questions – in particular, those associated with human pain – these techniques have yet to be widely adopted due to the technical challenges associated with implementing such methods, the lack of a broad expertise in optimal experimental design, and the requirement, in some cases, for bespoke, home-built instrumentation. Here we propose to overcome these challenges by constructing a spatial core that will initially focus on the spatially resolved transcriptomics approach MERFISH (multiplexed error robust fluorescence in situ hybridization). MERFISH is a massively multiplexed form of single-molecule fluorescence in situ hybridization that can image and identify hundreds to thousands (and more) different RNAs, and leads this field with its unique combination of sub-cellular resolution, near 100%-detection efficiency, low false-positive rate, low cost, and ultra- high throughput. MERFISH provides spatially resolved, single-cell expression profiles in millions of cells across tens of cm2 of tissues, revealing new insights from this unprecedented molecular and spatial resolution. This spatial core will be housed within the laboratory of Dr. Jeffrey Moffitt, a co-inventor of MERFISH. Only a few labs in the world are currently equipped to perform MERFISH, of which the Moffitt lab is one; the Moffitt lab has extensive experience with MERFISH in central-nervous-system tissues; and the Moffitt lab is continuing to advance the capabilities of this technology. For these reasons, our spatial core, whose staff will be embedded in the Moffitt lab, should be well positioned to leverage this novel technology, to integrate new innovations in this space, and to deploy, as needed, emerging new spatial techniques with increased performance. The spatial core will provide a start-to-finish service that coordinates all aspects of the design, execution, quality control, and interpretation of spatial measurements, which we will supplement as needed with an array- based, capture method (Visium). Key services will involve consultation on the design of targeted gene panels; the design and production of probe libraries; the optimization of tissue preparation, slicing, and clearing methods; rapid RNA integrity measurements to aid in preparation and sample prioritization; MERFISH measurement on state-of-the-art, home-built equipment; multi-tier data analysis, including identification of RNA and cell boundaries; and, finally, the calculation and interpretation of quality control and quality assurance metrics embedded within each MERFISH measurement. Critically, not only will this core provide fundamental new insights into human pain-associated neurological processes in health and disease, it will also aid in the dissemination of spatial transcriptomics to other pain-associated centers and the broader scientific community, magnifying the broad scientific impact of our efforts here.