Cell-type-specific Brain Methylomes Profiled via Ultralow-input Microfluidics

Methylomic studies require substantial amounts of DNA samples and this restriction hinders applications involving scarce animal or patient samples with direct biomedical relevance. Here we report a microfluidics-based reduced representative bisulfite sequencing protocol, MIcrofluidic Diffusion-based RRBS (MID-RRBS), that permits methylomic profiling with sub-1 ng starting DNA. Using this technology, we studied DNA methylation in NeuN+ and NeuN- fractions isolated from mouse cerebellum, revealing cell-type specific methylomic patterns. We also studied the DNA methylation in NeuN+ nuclei isolated from clozapine or vehicle treated mouse frontal cortex. We examined genome-wide DNA methylation profiles of GM12878 cell line (with starting DNA sample amounts in the range of 0.3-10 ng and of single cells), NeuN+ and NeuN- fractions from mouse cerebellum (with starting DNA sample amounts of 0.5 or 10 ng) using MID-RRBS. We generated RNA-seq data on NeuN+ and NeuN- nuclei from mouse cerebellum. We also included MID-RRBS and RNA-seq data obtained on homogenates from mouse cerebellum. Finally, we generated MID-RRBS and RNA-seq data on NeuN+ nuclei isolated from clozapine or vehicle treated mouse frontal cortex.甲基化组学（methylomic）研究需要大量DNA样本，这一限制阻碍了其在稀缺动物或具有直接生物医学相关性的患者样本相关应用中的开展。本研究报道了一种基于微流控（microfluidics）的简化代表性亚硫酸氢盐测序（reduced representative bisulfite sequencing, RRBS）方案——基于微流控扩散的RRBS（MIcrofluidic Diffusion-based RRBS, MID-RRBS），该方案可实现起始DNA量低于1 ng的甲基化组学分析。利用该技术，我们对从小鼠小脑分离得到的NeuN阳性（NeuN+）与NeuN阴性（NeuN-）组分中的DNA甲基化进行了分析，揭示了细胞类型特异性的甲基化组模式。我们还对从氯氮平（clozapine）或赋形剂（vehicle）处理的小鼠额叶皮层中分离得到的NeuN阳性细胞核的DNA甲基化进行了研究。我们利用MID-RRBS技术，对GM12878细胞系（起始DNA样本量为0.3~10 ng，且包含单细胞样本）、小鼠小脑来源的NeuN+与NeuN-组分（起始DNA样本量为0.5 ng或10 ng）的全基因组DNA甲基化谱进行了检测。我们获取了小鼠小脑来源的NeuN+与NeuN-细胞核的RNA测序（RNA-seq）数据。本数据集同时纳入了小鼠小脑匀浆的MID-RRBS与RNA-seq数据。最后，我们获取了从氯氮平或赋形剂处理的小鼠额叶皮层中分离得到的NeuN阳性细胞核的MID-RRBS与RNA-seq数据。