Spatial Core (Moffit)

空间分辨的单细胞转录组学技术是一种令人兴奋的新兴工具箱，用于创建 分子定义的、细胞解析的，并且通常是功能注释的组织图谱。在提供 对广泛的组织和问题的变革性新见解的潜力-特别是， 与人类疼痛相关-由于技术挑战，这些技术尚未被广泛采用 与实施这些方法相关，缺乏最佳实验设计方面的广泛专业知识，以及 在某些情况下，需要定制的自制仪器。 在这里，我们建议通过构建一个空间核心来克服这些挑战，这个空间核心最初将专注于 空间分辨转录组学方法MERFISH（多路错误鲁棒荧光原位 杂交）。MERFISH是单分子荧光原位杂交的大规模多重形式 它可以成像和识别数百到数千种（甚至更多）不同的RNA，并以其独特的优势引领这一领域。 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提供了空间分辨的，单细胞表达谱，在数百万个细胞中， 数十平方厘米的组织，揭示了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MERFISH的共同发明者Jeffrey Moffitt博士的实验室内。只 目前世界上有几个实验室配备了MERFISH，其中莫菲特实验室是其中之一;莫菲特实验室 实验室在MERFISH在中枢神经系统组织中有丰富的经验;莫菲特实验室正在继续 to advance推进the capabilities能力of this technology技术.基于这些原因，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，将新的创新整合到这个领域。 空间，并根据需要部署新出现的具有更高性能的空间技术。 空间核心将提供一个从头到尾的服务，协调设计，执行， 质量控制和空间测量的解释，我们将根据需要补充一个阵列- 捕获方法（Visium）。主要服务将包括就目标基因组的设计进行咨询; 探针库的设计和生产;组织制备、切片和清洁的优化 方法;快速RNA完整性测量，以帮助制备和样品优先化; MERFISH 在最先进的自制设备上进行测量;多层数据分析，包括RNA的鉴定 和细胞边界;最后，质量控制和质量保证的计算和解释 每个MERFISH测量中嵌入的指标。重要的是，这个核心不仅将提供基本的 对健康和疾病中人类疼痛相关神经过程的新见解，它还将有助于 将空间转录组学传播到其他疼痛相关中心和更广泛的科学界， 扩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.