Epigenetic tools and resources for cell-type and spatial analysis of individual mammalian non-neuronal cells

摘要 哺乳动物的大脑是一个极其复杂的器官，无数种细胞类型紧密地协同工作， 执行一系列复杂的任务，从运动功能到意识的存储和执行。这些 细胞类型广泛地分为神经元和非神经元分类，后者基本上 为电活动神经元提供支持系统和维持 成分随着单细胞平台的出现，我们现在有能力深入评估和 表征大脑中的所有细胞类型;然而，迄今为止的大多数研究都专门针对 神经元，在很大程度上忽略了它们的非神经元对应物。 在我们提出的研究计划中，我们将直接解决缺乏非神经元细胞的单细胞组学数据的问题。 通过部署我们开发的创新技术来分析表观遗传特性， 高吞吐量的单细胞水平。我们将特别关注人类的非神经元细胞， 通过富集NeuN（-）群体来研究啮齿动物的大脑，这是许多以神经元为重点的研究的倒数。 试验将包括通过部署染色质可及性试验评估调节元件的使用， DNA甲基化的全基因组分析，并评估染色体中染色质的三维折叠。 原子核除了在区域水平上分析单细胞外，我们还将调整我们的检测平台， 通过高密度区域二次抽样跟踪空间信息。我们将部署空间感知检测 在缺血性损伤的情况下，其导致神经胶质反应性从损伤辐射出的梯度 绝佳的价钱最后，我们对具有细胞类型和反应状态特异性的调节网络的评估非常适合 用于设计高度特异性的转基因报告小鼠。我们将使用我们确定的 驱动激活INTACT报告基因的标准和分裂重组酶构建体的调控模块 该系统能够快速有效地分离高纯度的靶细胞。我们建议的资源 发展将广泛地使数据的解释和研究的发展，针对非 哺乳动物大脑的神经元成分。

ABSTRACT The mammalian brain is an enormously complex organ with myriad cell types cohesively working together to carry out a host of intricate tasks, from motor functions, to the storing and execution of consciousness. These cell types broadly fall into neuronal and non-neuronal classifications, the latter of which substantially outnumber the former and provide the support system and maintenance for the electrically active neuronal component. With the advent of single-cell platforms, we now have the capability to deeply assess and characterize all cell types in the brain; however, the majority of studies to date have specifically targeted neurons, largely neglecting their non-neuronal counterpart. In our proposed plan of research, we will directly address the dearth of single-cell omics data on non-neuronal cell types by the deployment of innovative technologies we have developed to assay epigenetic properties at the single-cell level in high throughput. We will focus specifically on non-neuronal cells in the human and rodent brain by enriching for the NeuN(-) population, the reciprocal of numerous neuron-focused studies. Assays will include the assessment of regulatory element usage by deploying chromatin accessibility assays, genome-wide profiling of DNA methylation, and assessing the three-dimensional folding of chromatin in the nucleus. In addition to profiling single cells at the regional level, we will adapt our assay platform to include the tracking of spatial information by high-density regional subsampling. We will deploy the spatially aware assay variant in the context of ischemic injury, which results in a gradient of glial reactivity radiating out from the injury site. Lastly, our assessment of regulatory networks with cell type and reactive-state specificity is ideally suited for the design of highly specific transgenic reporter mice. We will produce these lines using our identified regulatory modules to drive standard and split recombinase constructs that activate the INTACT reporter system to enable rapid and efficient isolation of target cells with high purity. The resources we propose to develop will broadly enable the interpretation of data and the development of studies that target the non- neuronal component of the mammalian brain.