High-content single-cell epigenetic technologies scalable to the human brain

项目摘要 为了实现大脑倡议细胞普查网络编目和产生分子图谱的目标 人脑中的每一种细胞类型；单细胞分析吞吐量和 承保范围是必需的。在这份提案中，我们详细介绍了通过利用新的化学物质来满足这些需求的策略 工作流和组合索引技术。这将包括发展两个互补的 技术(S3和S4)，克服了当前技术的主要限制，生产出更高的 基于DNA的属性的覆盖范围，包括染色质可及性(S3-ATAC)和组蛋白标记(S4-CAT)。 这些技术还将被用来开发捕获RNA转录的分析方法 DNA编码的属性。我们还将扩展这些工作流，以实现数量级的改进 我们之前描述的高通量分析单细胞DNA甲基化的技术的细胞覆盖率 (SCI-MET)。覆盖范围的扩大还将使针对池化单细胞的定向捕获的开发成为可能 DNA甲基化文库，以降低每个细胞的测序成本，同时分析感兴趣的区域。这些捕获 文库将支持更大的细胞数据集，这些数据集可以补充来自 较小的细胞样本。最后，我们将通过制作一份初步的地图集来演示这些技术 人类初级视觉皮质和海马体中的表观遗传学特征作为 BICCN的大规模协调努力。

PROJECT ABSTRACT To meet the goal of the BRAIN Initiative Cell Census Network to catalogue and produce molecular profiles of every cell type in the human brain; order-of-magnitude improvements in single-cell assay throughput and coverage are required. In this proposal, we detail strategies to meet these needs by leveraging novel chemistry workflows and combinatorial indexing techniques. This will include the development of two complementary techniques (s3 and s4) that overcome the major limitations of current technologies to produce far higher coverage of DNA-based properties, including chromatin accessibility (s3-ATAC) and histone marks (s4-CAT). These technologies will also be leveraged to develop assays that capture RNA transcription alongside the DNA-encoded property. We will also extend these workflows to produce order-of-magnitude improvements in cell coverage for our previously-described technology to profile single-cell DNA methylation in high throughput (sci-MET). The improved coverage will also enable the development of targeted capture for pooled single-cell DNA methylation libraries to reduce sequencing costs per cell while profiling regions of interest. These capture libraries will enable much larger cell datasets that can complement whole-genome single-cell profiles from a smaller sampling of cells. Finally, we will demonstrate these technologies by producing a preliminary atlas of epigenetic profiles in the human primary visual cortex and hippocampus to serve as a starting point for the large scale coordinated efforts of the BICCN.