Department of Biological Science, Program in Neuroscience, Florida State University, Tallahassee, FL 32306, USA.
Genes (Basel). 2022 Feb 6;13(2):306. doi: 10.3390/genes13020306.
DNA methylation plays essential roles in various cellular processes. Next-generation sequencing has enabled us to study the functional implication of DNA methylation across the whole genome. However, this approach usually requires a substantial amount of genomic DNA, which limits its application to defined cell types within a discrete brain region. Here, we applied two separate protocols, Accel-NGS Methyl-Seq (AM-seq) and Enzymatic Methyl-seq (EM-seq), to profile the methylome of D2 dopamine receptor-expressing medium spiny neurons (D2-MSNs) in mouse nucleus accumbens (NAc). Using 40 ng DNA extracted from FACS-isolated D2-MSNs, we found that both methods yielded comparably high-quality methylome data. Additionally, we identified numerous unmethylated regions (UMRs) as cell type-specific regulatory regions. By comparing the NAc D2-MSN methylome with the published methylomes of mouse prefrontal cortex excitatory neurons and neural progenitor cells (NPCs), we identified numerous differentially methylated CpG and non-CpG regions. Our study not only presents a comparison of these two low-input DNA whole genome methylation profiling protocols, but also provides a resource of DNA methylome of mouse accumbal D2-MSNs, a neuron type that has critical roles in addiction and other neuropsychiatric disorders.
DNA 甲基化在各种细胞过程中发挥着重要作用。下一代测序技术使我们能够研究整个基因组中 DNA 甲基化的功能意义。然而，这种方法通常需要大量的基因组 DNA，这限制了它在离散脑区中特定细胞类型中的应用。在这里，我们应用了两种分离的方案，加速基因组测序甲基化测序（Accel-NGS Methyl-Seq，AM-seq）和酶促甲基化测序（Enzymatic Methyl-seq，EM-seq），来分析小鼠伏隔核（NAc）中表达 D2 多巴胺受体的中型棘突神经元（D2-MSNs）的甲基组。使用从 FACS 分离的 D2-MSN 中提取的 40ng DNA，我们发现这两种方法都产生了质量相当高的甲基组数据。此外，我们还鉴定了许多未甲基化区域（UMRs）作为细胞类型特异性调控区域。通过比较 NAc D2-MSN 的甲基组与已发表的小鼠前额叶皮层兴奋性神经元和神经祖细胞（NPCs）的甲基组，我们鉴定了许多差异甲基化的 CpG 和非 CpG 区域。我们的研究不仅比较了这两种低输入 DNA 全基因组甲基化分析方案，还提供了小鼠伏隔核 D2-MSN 的 DNA 甲基组资源，D2-MSN 是一种在成瘾和其他神经精神疾病中具有关键作用的神经元类型。
