Ma Sai, de la Fuente Revenga Mario, Sun Zhixiong, Sun Chen, Murphy Travis W, Xie Hehuang, González-Maeso Javier, Lu Chang
Department of Biomedical Engineering and Mechanics, Virginia Tech, Blacksburg, VA 24061, USA.
Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USA.
Nat Biomed Eng. 2018 Mar;2(3):183-194. Epub 2018 Mar 7.
Methylomic analyses typically require substantial amounts of DNA, thus hindering studies involving scarce samples. Here, we show that microfluidic diffusion-based reduced representative bisulfite sequencing (MID-RRBS) permits high-quality methylomic profiling with nanogram-to-single-cell quantities of starting DNA. We used the microfluidic device, which allows for efficient bisulfite conversion with high DNA recovery, to analyse genome-wide DNA methylation in cell nuclei isolated from mouse brains and sorted into NeuN+ (primarily neuronal) and NeuN- (primarily glial) fractions, and to establish cell-type-specific methylomes. Genome-wide methylation and methylation in low-CpG-density promoter regions showed distinct patterns for NeuN+ and NeuN- fractions from the mouse cerebellum. The identification of substantial variations in the methylomic landscapes of the NeuN+ fraction of the frontal cortex of mice chronically treated with an atypical antipsychotic drug suggests that this technology can be broadly used for cell-type-specific drug profiling and for the study of drug-methylome interactions.
甲基化组分析通常需要大量的DNA，因此阻碍了涉及稀缺样本的研究。在这里，我们表明基于微流控扩散的简化代表性亚硫酸氢盐测序（MID-RRBS）允许使用纳克级到单细胞量的起始DNA进行高质量的甲基化组分析。我们使用了这种微流控装置，它能够实现高效的亚硫酸氢盐转化并具有高DNA回收率，用于分析从小鼠大脑中分离并分选到NeuN+（主要是神经元）和NeuN-（主要是神经胶质）组分中的细胞核的全基因组DNA甲基化，并建立细胞类型特异性甲基化组。来自小鼠小脑的NeuN+和NeuN-组分在全基因组甲基化和低CpG密度启动子区域的甲基化表现出不同的模式。对长期用非典型抗精神病药物治疗的小鼠额叶皮质NeuN+组分的甲基化组图谱中大量差异的鉴定表明，该技术可广泛用于细胞类型特异性药物分析以及药物-甲基化组相互作用的研究。
