scNMT-seq of the adult NSC lineage - RNA

Stem cells in the adult brain are specialized astrocytes capable of generating neurons and glial cells. While neural stem cells (NSCs) and common astrocytes have clearly distinct functions, they share highly similar transcriptome profiles. How stemness is molecularly encoded is therefore unclear. Here we use single-cell NMT-seq to simultaneously characterize the transcriptome, DNA methylome and chromatin accessibility of astrocytes and the NSC lineage in the healthy and ischemic brain. Our data reveal distinct methylation profiles associated with either astrocyte or stem cell function. Stemness is conferred by methylation of astrocyte genes and demethylation of neurogenic genes that are expressed only later. Surprisingly, ischemic injury unlocks the stemness-methylome in common astrocytes enabling generation of neuroblasts. Furthermore, we show that oligodendrocytes employ Tet-mediated demethylation to regulate expression of myelin-related genes, many of which are abnormally methylated in multiple sclerosis. Overall, we show that DNA methylation is a promising target for regenerative medicine. Astrocytes, oligodendrocytes, adult neural stem cells as well as their progeny were isolated via flourescence-activated cell sorting (FACS) from the ventricular-subventricular zone, striatum, and olfactory bulb. Each biological replicate (pA, pB, …) consists of one or more mice. Cells were subjected to scNMT-seq in order to quantify DNA methylation, chromatin accessibility, and gene expression at single cell resolution.

成体大脑内的干细胞为特化星形胶质细胞，具备分化生成神经元与神经胶质细胞的能力。尽管神经干细胞（neural stem cells, NSCs）与普通星形胶质细胞功能迥异，但二者的转录组谱却高度相似，因此干细胞干性的分子编码机制至今仍不明确。本研究采用单细胞NMT测序（single-cell NMT-seq）技术，同时表征健康脑组织与缺血脑组织中星形胶质细胞及神经干细胞谱系的转录组、DNA甲基化组与染色质可及性。研究数据显示，与星形胶质细胞功能或干细胞干性相关的甲基化谱存在显著差异：干细胞干性的维持依赖于星形胶质细胞基因的甲基化，以及仅在后续阶段表达的神经发生相关基因的去甲基化。令人意外的是，缺血性损伤可激活普通星形胶质细胞内的干性甲基化组，使其具备生成成神经细胞的能力。此外，本研究发现少突胶质细胞通过Tet蛋白介导的去甲基化调控髓鞘相关基因的表达，其中诸多基因在多发性硬化患者体内存在异常甲基化。综上，本研究证实DNA甲基化可作为再生医学的潜在干预靶点。研究人员通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）技术，从脑室-室管膜下区、纹状体与嗅球中分离得到星形胶质细胞、少突胶质细胞、成体神经干细胞及其子代细胞。每一个生物学重复样本（pA、pB等）由一只或多只小鼠的组织构成。随后对分离得到的细胞进行单细胞NMT测序（scNMT-seq），以在单细胞分辨率下量化DNA甲基化水平、染色质可及性与基因表达情况。