Transcriptome & Methylome Analysis of Single Cells

转录组

• 批准号: 7990042
• 负责人: SHERMAN Morton WEISSMAN
• 金额: $ 24.83万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-25 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7990042
• 关键词: Achievement; Automobile Driving; Cell Count; Cell Differentiation process; Cells; Child Development; Chromosomes; Clinical; Complementary DNA; Complex; CpG Islands; Cytolysis; DNA; DNA Methylation; DNA Modification Process; Development; Developmental Disabilities; Diagnosis; Discrimination; Disease; Elements; Embryo; Embryonic Development; Epigenetic Process; Event; Excision; Future; Gene Expression; Gene Expression Profile; Generations; Genome; Genomics; Human Development; Immune system; Length; Malignant Neoplasms; Messenger RNA; Methods; Methylation; Modeling; Molecular; Molecular Conformation; Morula; Mus; Neurons; Pattern; Procedures; Protein Isoforms; Psyche structure; RNA Splicing; Regulation; Research; Sequence Analysis; Single-Stranded DNA; Site; Staging; Surveys; Technology; Testing; Transcript; Tube; Work; blastocyst; chromatin immunoprecipitation; critical period; design; epigenomics; experience; genome wide association study; genome-wide; human disease; insight; novel strategies; oligo (dT); preimplantation; public health relevance; single cell analysis; tool; transcriptomics

DESCRIPTION (provided by applicant): Robust technologies are lacking for genome-wide profiling of single cell transcriptomic and epigenomic changes. This situation constitutes a bottleneck and hinders the progress of many important fields where single cell analysis is required. Our recently developed procedure, whole DNA pool amplification (WPA), offers highly specific amplification of a complex DNA mixture with high efficiency and minimized bias. WPA can amplify as little as sub-femtogram quantities of DNA, generating micrograms of specific product. Recently we have adapted WPA toward developing a method for whole transcriptome amplification of the entire mRNA transcript profile at full lengths and with strandedness, and preliminarily tested it on 1 nanogram of cDNA, 10 cells and single cells. In this proposal, using single cells isolated from different preimplantation stage embryos as a proof of principle model, we will focus on the development of single cell research tools for genome-wide quantitative assessment of mRNA transcriptome and CpG methylation by deep sequencing analysis. Specifically, we plan to: 1). optimize a procedure for cell lysis, gDNA removal, and cDNA generation from single cells; 2). develop an approach for generating highly specific materials suitable for high throughput sequencing by circularizing first strand cDNA from the intact cells treated as above, amplifying this cDNA in full lengths and followed by olig-dT/rU selection when necessary; 3). develop a procedure that combines enzymatic discrimination of DNA methylation, DNA circularization, and isolation of CpG-rich DNA fragments, which will enable high-throughput sequencing to identify differentially methylated HpaII or other restriction sites for a single cell; and finally 4). analyze the mRNA transcriptome (covering expression, splicing form, allelic specific expression, etc) and CpG methylation patterns of 3 types of single cells isolated from mouse preimplantation embryos. This study will enable us to study the genome-wide changes of gene expression (mRNA), DNA methylation, and in the future other epigenomic elements, at single cell level and at 4-dimmersions for preimplantation embryos, and allow for unprecedented characterization of this critical period of development and differentiation. The technological and theoretical achievements from this project will contribute significantly to the study of the mechanism for human development and diseases in terms of its epigenomic regulation, and will eventually benefit the understanding, diagnosis and treatment of human diseases such as those related to children development, cancer, neuron/mental, and immune system. PUBLIC HEALTH RELEVANCE: The technological and theoretical achievements from this project will contribute significantly to the study of the mechanism for human development and diseases in terms of its epigenomic regulation, and will eventually benefit the understanding, diagnosis and treatment of human diseases such as children development disorders, cancer, neuron/mental, and immune system related clinical problems.

描述（由申请人提供）：缺乏用于单细胞转录组和表观基因组变化的全基因组分析的稳健技术。这种情况构成了瓶颈，阻碍了许多需要单细胞分析的重要领域的进展。我们最近开发的程序，全DNA池扩增（WPA），提供了一个复杂的DNA混合物的高效率和最小化的偏见高度特异性扩增。WPA可以扩增低至毫微微克的DNA，产生微克的特定产物。最近，我们已经适应WPA发展的方法，在全长和链的整个mRNA转录谱的全转录组扩增，并初步测试了1纳克的cDNA，10个细胞和单细胞。在这项提案中，使用从不同植入前阶段胚胎中分离的单细胞作为原理模型的证明，我们将专注于开发单细胞研究工具，通过深度测序分析对mRNA转录组和CpG甲基化进行全基因组定量评估。具体而言，我们计划：1）。优化用于细胞裂解、gDNA去除和从单细胞产生cDNA的程序; 2）.开发一种用于产生适合于高通量测序的高度特异性材料的方法，所述方法通过环化来自如上处理的完整细胞的第一链cDNA，扩增全长的该cDNA，然后在必要时进行olig-dT/rU选择; 3）.开发一种结合DNA甲基化、DNA环化和富含CpG的DNA片段的分离的酶促鉴别的程序，这将使得高通量测序能够鉴定单个细胞的差异甲基化的HpaII或其他限制性位点;以及最后4）。分析小鼠植入前胚胎3种单细胞的mRNA转录组（覆盖表达、剪接形式、等位基因特异性表达等）和CpG甲基化模式。这项研究将使我们能够研究基因表达（mRNA），DNA甲基化的全基因组变化，并在未来的其他表观基因组元素，在单细胞水平和4-dimmersions的植入前胚胎，并允许前所未有的表征这一关键时期的发展和分化。本项目的技术和理论成果将对人类发育和疾病的表观基因调控机制的研究做出重要贡献，最终将有利于儿童发育、癌症、神经/精神、免疫系统等人类疾病的理解、诊断和治疗。 公共卫生关系：该项目的技术和理论成果将有助于从表观基因组调控的角度研究人类发育和疾病的机制，并最终有利于儿童发育障碍、癌症、神经元/精神和免疫系统相关临床问题等人类疾病的理解、诊断和治疗。