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，以及 非编码RNA的表达。DNA甲基化是目前研究最深入的表观遗传修饰 哺乳动物。全基因组DNA甲基化图谱(即DNA甲基组)被建立 在发育的早期调节和干扰的甲基组在疾病中起关键作用 发展。已经有许多全基因组技术被开发用于图谱分析 DNA甲基组。全基因组亚硫酸盐测序(WGBS)通常被认为是 DNA甲基化分析的金标准。该方法生成具有以下特征的甲基组图谱 单核苷酸拆分。传统的WGBS需要大量的DNA。虽然 单细胞亚硫酸盐测序是最近被证明的，这些分析在很大程度上是基于 在管子/井板上的操作和手工程序，使大量的快速加工 单细胞数量(&gt；1000)困难。在这个项目中，我们将开发一种高通量的单 基于微流控液滴的细胞亚硫酸盐测序技术，称为Drop-BS 站台。我们将通过检查肿瘤内的异质性来测试和验证这项技术 乳腺肿瘤患者和患者来源异种移植(PDX)的单细胞DNA甲基组 老鼠模型。

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.