Drop-BS: high-throughput single-cell bisulfite sequencing on a microfluidic droplet platform

项目概要 细胞过程受到不同表观遗传层之间复杂的相互作用的调节 信息，包括 DNA 甲基化、组蛋白修饰、核小体位置和 非编码RNA的表达。 DNA甲基化是研究最充分的表观遗传修饰 哺乳动物。建立全基因组 DNA 甲基化谱（即 DNA 甲基化组） 处于发育早期的调控和受干扰的甲基化组与疾病密切相关 发展。已经开发了许多用于分析的全基因组技术 DNA 甲基化组。全基因组亚硫酸氢盐测序（WGBS）通常被认为是 DNA 甲基化分析的金标准。该方法生成甲基组谱 单核苷酸分辨率。传统的 WGBS 需要大量 DNA。虽然 最近展示了单细胞亚硫酸氢盐测序，这些测定很大程度上基于 管/孔板中的操作和手动程序，使得快速处理大量 单细胞数量（>1000）困难。在这个项目中，我们将开发一种高通量单 基于微流控液滴的细胞亚硫酸氢盐测序技术，简称Drop-BS 平台。我们将通过检查肿瘤内异质性来测试和验证该技术 来自患者和患者来源的异种移植物 (PDX) 的乳腺肿瘤的单细胞 DNA 甲基化组 鼠标模型。

Project Summary Cellular processes are regulated by a complex interplay among different layers of epigenetic information, including DNA methylation, histone modification, nucleosome position, and expression of noncoding RNA. DNA methylation is the best studied epigenetic modification in mammals. The genome-wide DNA methylation profile (i.e., DNA methylome) is established early in development for regulation and perturbed methylome is critically involved in disease development. There have been a number of genome-wide technologies developed for profiling DNA methylomes. Whole genome bisulfite sequencing (WGBS) is generally considered the gold standard for DNA methylation analyses. The approach generates methylomic profile with single-nucleotide resolution. Conventional WGBS requires a large amount of DNA. Although single-cell bisulfite sequencing was demonstrated recently, these assays were based heavily on manipulation in tubes/well plates and manual procedures, making rapid processing of a large number (>1000) of single cells difficult. In this project, we will develop a high-throughput single- cell bisulfite sequencing technology, referred to as Drop-BS, based on a microfluidic droplet platform. We will test and validate the technology by examining intratumoral heterogeneity in single-cell DNA methylomes of breast tumors from patients and patient-derived xenograft (PDX) mouse model.