A Single-Cell Transcriptome Atlas for Zebrafish Development

问题：不同的疾病会影响不同的细胞类型，每种类型都由其独特的表达方式来定义。 基因：它的转录组。单细胞转录组测序（scRNA-seq）是鉴定 通过识别共享相似转录组的细胞簇，在健康和疾病中检测细胞类型。scRNA- 然而，seq方法通常分离样品，因此丢失了关于每个细胞在哪里的信息。 集群驻留在主体中。为了了解每一个集群在一个完整的动物，最好的电流 方法是对整个标本或组织切片进行原位杂交。问题是传统的原地 杂交方案很少达到足够的分辨率来定位体内的稀有细胞类型。的 解决方案是单分子荧光原位杂交（smFISH），一种检测和计数 细胞内特定的RNA分子。 此管理补充申请的具体目的是购买Keyence BZ-X800 E 荧光显微镜，使smFISH实验。该设备将更有效地实现 在受资助项目范围内的母赠款的目的。拟议工作不与 资助的工作，因为我们不能有效地执行smFISH没有要求的 设备. 这一补充申请符合父母补助金的主要目标： 斑马鱼的转录组图谱，并通过原位杂交验证聚类鉴定。商业上 可用的smFISH试剂盒最近已经上市，但我们目前的荧光显微镜是 不适合smFISH。相比之下，基恩士荧光显微镜可以有效地验证scRNA- 通过smFISH测序细胞簇。它结合了一个正式的显微镜，一个读板器，一个载玻片扫描仪， 和一个共聚焦荧光显微镜，这将使验证实验对斑马鱼不可能 我们现有的设备。 要求的设备将影响NIH研究所和中心的全方位，因为我们执行 scRNA-seq和smFISH在完整动物上涵盖所有器官系统。受影响的研究领域 包括遗传学、发育生物学、细胞生物学和病理学。完成的时间轴 包括立即购买的显微镜获奖通知，交付两个星期后， 直接通过smFISH实验。所有资金将在本期内使用。所需设备将 通过在初次实验动物体内定位细胞类型来改善动物模型的重要资源 人类疾病模型，从而提高健康和疾病的细胞机制的知识。

The Problem: Different diseases affect different cell types, each defined by its unique set of expressed genes: its transcriptome. Single cell transcriptome sequencing (scRNA-seq) is a powerful way to identify cell types in health and disease by identifying clusters of cells that share similar transcriptomes. scRNA- seq methods, however, generally dissociate samples, thus losing information about where each cell cluster resides in the body. To learn where each cluster resides in an intact animal, the best current method is in situ hybridization to whole mounts or tissue sections. The problem is that traditional in situ hybridization protocols seldom achieve sufficient resolution to locate rare cell types in the body. The solution is Single molecule Fluorescent In Situ Hybridization (smFISH), a method to detect and count specific individual RNA molecules inside cells. The Specific Aim of this Administrative Supplement request is to purchase a Keyence BZ-X800E fluorescence microscope to enable smFISH experiments. This equipment will more effectively achieve the aims of the parent grant within the scope of the funded project. Proposed work does not overlap with funded work in the parent award because we cannot perform smFISH effectively without the requested equipment. This supplement request aligns with the parent grant’s main goals: to develop a single-cell transcriptomic atlas for zebrafish and to validate cluster identification by in situ hybridization. Commercially available smFISH kits have recently become available, but our current fluorescence microscope is insufficient for smFISH. In contrast, the Keyence fluorescence microscope can efficiently validate scRNA- seq cell clusters by smFISH. It combines features of a formal microscope, a plate reader, a slide scanner, and a confocal fluorescence microscope that will enable validation experiments on zebrafish not possible with our current equipment. Requested equipment will impact the full range of NIH Institutes and Centers because we perform scRNA-seq and smFISH on intact animals encompassing all organ systems. Affected research fields include genetics, developmental biology, cell biology, and pathology. The timeline for completion includes immediate purchase of the microscope on award notification, delivery two weeks later, followed directly by smFISH experiments. All funds will be spent in the current period. Requested equipment will improve an important resource for animal models by localizing cell types in the body of a premiere animal model of human disease, thus enhancing knowledge of cellular mechanisms in health and disease.