Transcriptome & Methylome Analysis of Single Cells

描述（由申请人提供）：缺乏对单细胞转录组和表观基因组变化进行全基因组分析的强大技术。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），提供高特异性的复杂DNA混合物的扩增，具有高效率和最小的偏差。WPA可以扩增到亚飞图数量的DNA，产生微克的特定产物。最近，我们将WPA技术应用于开发一种全长和带链的全转录组扩增方法，并在1纳克cDNA、10个细胞和单细胞上进行了初步测试。在本提案中，我们将利用从不同着床前阶段胚胎中分离的单细胞作为原理模型，重点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)优化细胞裂解，gDNA去除和单细胞cDNA生成的程序；2).开发一种方法，通过从上述处理的完整细胞中循环第一链cDNA，扩增该cDNA的全长，并在必要时进行oligo - dt /rU选择，生成适合高通量测序的高特异性材料；3)开发一种结合DNA甲基化，DNA环化和富含cpg的DNA片段分离的酶促鉴别的程序，这将使高通量测序能够识别单个细胞的差异甲基化HpaII或其他限制性位点；4)分析小鼠着床前胚胎分离的3种单细胞的mRNA转录组（包括表达、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在单细胞水平和4维空间研究植入前胚胎基因表达（mRNA）、DNA甲基化以及其他表观基因组元素的全基因组变化，并允许对这一发育和分化的关键时期进行前所未有的表征。本项目的技术和理论成果将为研究人类发育和疾病的表观基因组调控机制做出重要贡献，并最终有助于对儿童发育、癌症、神经/精神和免疫系统等人类疾病的认识、诊断和治疗。

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.