Spatial Core (Moffit)

空间分辨的单细胞转录切割技术是一种令人兴奋的新兴工具箱，用于创建 分子定义的，细胞分辨的，通常还有功能注释的组织图谱。在提供 对广泛的组织和问题有变革性的新见解的潜力--特别是那些 与人类疼痛相关-由于技术上的挑战，这些技术尚未被广泛采用 与实施这些方法相关，在最佳试验设计方面缺乏广泛的专业知识，以及 在某些情况下，对定制的自制仪器的要求。 在这里，我们建议通过构建空间核心来克服这些挑战，该空间核心最初将专注于 MERFISH(多路误差稳健原位荧光)空间分辨转录组学方法 杂交)。水星鱼是一种大规模复合形式的单分子荧光原位杂交 它可以成像和识别成百上千(甚至更多)不同的RNA，并以其独特的 结合亚细胞分辨率、接近100%的检测效率、低假阳性率、低成本和超 高吞吐量。MerFish在数百万个细胞中提供空间分辨的单细胞表达谱 几十平方厘米的组织，揭示了这一前所未有的分子和空间分辨率的新见解。 这个空间核心将被安置在杰弗里·莫菲特博士的实验室里，他是MerFish的共同发明者。仅限 世界上目前有几个实验室配备了进行MerFish实验的设备，莫菲特实验室就是其中之一； 实验室对中枢神经系统组织中的水鱼有丰富的经验；莫菲特实验室仍在继续 来提升这项技术的能力。出于这些原因，我们的空间核心，其工作人员将嵌入 在莫菲特实验室，应该能够很好地利用这项新技术，将新的创新整合到 并根据需要部署性能更高的新兴空间技术。 空间核心将提供从开始到结束的服务，协调设计、执行、 质量控制和空间测量的解释，我们将根据需要使用阵列进行补充- 基于捕获方法(维西姆)。主要服务将包括就靶向基因面板的设计提供咨询； 探针库的设计和生产；组织制备、切片和清除的优化 方法.辅助制备和样品优先顺序的快速核糖核酸完整性测量.墨鱼 对最先进的自制设备进行测量；多层数据分析，包括识别RNA 和单元格边界；最后，质量控制和质量保证的计算和解释 嵌入在每个MerFish测量中的指标。关键的是，这个核心不仅将提供基本的 对人类健康和疾病中与疼痛相关的神经过程的新见解，也将有助于 向其他疼痛相关中心和更广泛的科学界传播空间转录本， 放大了我们在这里的努力的广泛的科学影响。

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.