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

项目摘要 细胞过程受到不同层表观遗传学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， 非编码RNA的表达。DNA甲基化是研究最多的表观遗传修饰， 哺乳动物全基因组DNA甲基化谱（即，DNA甲基化）建立 在发育的早期进行调节，而甲基化组的紊乱与疾病密切相关。 发展已经开发了许多用于分析的全基因组技术 DNA甲基化。全基因组亚硫酸氢盐测序（WGBS）通常被认为是最好的测序方法。 DNA甲基化分析的黄金标准。该方法生成甲基化图谱， 单核苷酸分辨率。传统的WGBS需要大量的DNA。虽然 最近证实了单细胞亚硫酸氢盐测序，这些测定在很大程度上基于 操作管/孔板和手动程序，使快速处理大的 单个细胞数量（>1000）困难。在这个项目中，我们将开发一个高通量的单- 细胞亚硫酸氢盐测序技术，称为Drop-BS，基于微流体液滴 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 小鼠模型

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.