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.
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.
|Pan, Xinghua; Durrett, Russell E; Zhu, Haiying et al. (2013) Two methods for full-length RNA sequencing for low quantities of cells and single cells. Proc Natl Acad Sci U S A 110:594-9|
|Wang, Fang; Pan, Xinghua; Kalmbach, Keri et al. (2013) Robust measurement of telomere length in single cells. Proc Natl Acad Sci U S A 110:E1906-12|