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.

甲基组学研究（Methylomic studies）需要大量DNA样本，该限制条件极大阻碍了针对珍稀动物样本及具有直接生物医学相关性的患者样本的研究应用。本研究报道了一种基于微流控技术的简化代表性亚硫酸氢盐测序方案，即微流控扩散型简化代表性亚硫酸氢盐测序（MIcrofluidic Diffusion-based RRBS, MID-RRBS），该方案可实现起始DNA量低于1 ng的甲基组学分析。利用该技术，我们对从小鼠小脑分离的NeuN阳性与NeuN阴性组分开展了DNA甲基化分析，揭示了细胞类型特异性的甲基组模式。我们还对经氯氮平或溶剂对照处理的小鼠额叶皮层分离的NeuN阳性细胞核进行了DNA甲基化分析。我们使用MID-RRBS分析了GM12878细胞系（起始DNA样本量范围为0.3~10 ng，且包含单细胞样本）、小鼠小脑的NeuN阳性与NeuN阴性组分（起始DNA样本量为0.5或10 ng）的全基因组DNA甲基化谱。我们生成了小鼠小脑NeuN阳性与NeuN阴性细胞核的RNA测序（RNA-seq）数据。我们还纳入了从小鼠小脑匀浆获得的MID-RRBS与RNA-seq数据。最后，我们还生成了经氯氮平或溶剂对照处理的小鼠额叶皮层分离的NeuN阳性细胞核的MID-RRBS与RNA-seq数据。