Slide-seq: high-resolution in situ expression profiling for neuropathology

摘要 人脑标本是为数不多的直接研究分子的机会之一。 神经精神疾病的基础。基因和蛋白质的原位表达分析已经发生了革命性的变化 我们对许多这些疾病的了解，但尽管有这些进步，对于许多大脑疾病来说，组织 人们对引起临床症状的变化知之甚少。确定全套分子 变化(S)和每个变化所在的细胞类型(S)将通知用于研究的实验室模型 这些疾病，并为潜在的新治疗方法提供了假设。这份提案概述了一项新技术， Slide-Seq，能够在单细胞(~7)上对完整的脑组织切片进行全转录组分析 μm)分辨率。该方法应用了我最近开发的一种新的DNA条形码方案，该方案基于 分裂和池DNA合成，以构建装饰有高度多样性的珠子阵列的显微镜载玻片 记录每个基因的精细空间坐标，整个转录组。在本提案中，我们：(1)描述 将RNA从组织切片转移到细胞上所需的分子、计算和细胞生物学创新 这一阵列，并成功地报告了它们的空间位置；(2)明确列出了三种核心分析方法 衡量每一关键技术步骤的成功；(3)批判性地评估幻灯片的单元格分辨率- (4)将Slide-Seq应用于阿尔茨海默氏症小鼠模型 疾病以揭示周围细胞群体在早期对神经元淀粉样蛋白堆积的反应 疾病的各个阶段。总之，这些实验将产生一个经过充分验证的工具，具有广泛的适用性 生物学中的问题，尤其是对病理性脑组织的分析。

ABSTRACT Human brain specimens are one of few available opportunities to directly investigate the molecular underpinnings of neuropsychiatric diseases. Assays of gene and protein expression in situ have revolutionized our understanding of many of these diseases, but despite these advances, for many brain disorders, the tissue changes that give rise to clinical symptoms are poorly understood. Ascertaining the full set of molecular change(s) and the cell type(s) in which each change resides would inform the laboratory models used to study these illnesses, and offer hypotheses for potential new treatments. This proposal outlines a novel technology, Slide-seq, capable of performing whole-transcriptome analysis of intact brain tissue sections at single-cell (~7 μm) resolution. The method applies my recent development of a novel DNA barcoding scheme, based upon split-and-pool DNA synthesis, to construct a microscope slide adorned with a high-diversity bead array capable of recording fine spatial coordinates for each gene, transcriptome-wide. In this proposal, we: (1) describe the molecular, computational, and cell biological innovations necessary to transfer RNA from tissue sections onto this array, and successfully report their spatial positions; (2) outline three core assays for unambiguously measuring the success of each crucial technical step; (3) critically evaluate the single-cell resolution of Slide- seq by profiling the mouse adult hippocampus; and (4) apply Slide-seq to a mouse model of Alzheimer's disease to uncover how surrounding cell populations respond to neuronal amyloid accumulation in the early stages of disease. Together, these experiments will produce a well-validated tool with broad applicability to problems in biology, most especially the analysis of pathological brain tissue.