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

Drop-BS：微流控液滴平台上的高通量单细胞亚硫酸氢盐测序

基本信息

批准号：
9789912
负责人：
Chang Lu
金额：
$ 24.29万
依托单位：
VIRGINIA POLYTECHNIC INST AND ST UNIV
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-21 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9789912
关键词：
Biological Assay Breast Cancer Patient Cell Line Cell physiology Cells Complex Cytolysis DNA DNA Methylation Data Data Set Development Devices Disease Drops Epigenetic Process Future Genetic Fingerprintings Genome Genomics Gold Human Libraries Mammals Mammary Neoplasms Manuals Microfluidic Microchips Microfluidics Modification Nucleosomes Nucleotides Patients Population Positioning Attribute Preparation Procedures Protocols documentation Reaction Regulation Research Personnel Resolution Running Sampling Technology Testing Time Trees Tube Untranslated RNA Xenograft procedure base bisulfite bisulfite sequencing cost epigenomics forest genome-wide histone modification innovation interest malignant breast neoplasm methylome methylomics molecular subtypes mouse model next generation sequencing tool tumor tumor heterogeneity whole genome

项目摘要

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.

项目摘要细胞过程受到不同表观遗传层之间的复杂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甲基化是最佳研究的表观遗传修饰哺乳动物。建立了全基因组DNA甲基化谱（即DNA甲基团）在调节和扰动甲基的开发初期，疾病与疾病至关重要发展。已经开发了许多用于分析的全基因组技术 DNA甲基组。整个基因组硫酸盐测序（WGB）通常被认为是 DNA甲基化分析的金标准。该方法与单核苷酸分辨率。常规WGB需要大量DNA。虽然最近证明了单细胞亚硫酸盐测序，这些测定基于管子/井板和手动程序中的操作，快速处理大型单细胞的数字（> 1000）困难。在这个项目中，我们将开发一个高通量单一的基于微流体液滴的细胞亚硫酸盐测序技术，称为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的乳腺肿瘤的单细胞DNA甲基甲基和患者衍生的异种移植物（PDX）鼠标模型。