Integration of genome-wide approaches identifies lncRNAs of adult neural stem cells and their progeny in vivo [expression]

Long noncoding RNAs (lncRNAs) have been described in cell lines and various whole tissues, but lncRNA analysis of development in vivo is limited. Here, we comprehensively analyze lncRNA expression for the adult mouse subventricular zone neural stem cell lineage. We utilize complementary genome-wide techniques including RNA-seq, RNA CaptureSeq, and ChIP-seq to associate specific lncRNAs with neural cell types, developmental processes, and human disease states. By integrating data from chromatin state maps, custom microarrays, and FACS purification of the subventricular zone lineage, we stringently identify lncRNAs with potential roles in adult neurogenesis. shRNA-mediated knockdown of two such lncRNAs, Six3os and Dlx1as, indicate roles for lncRNAs in the glial-neuronal lineage specification of multipotent adult stem cells. Our data and workflow thus provide a uniquely coherent in vivo lncRNA analysis and form the foundation of a user-friendly online resource for the study of lncRNAs in development and disease. SVZ monolayer cultures were differentiated in vitro for 1, 2, 4 days, and gene expression changes were measured. SVZ lineage was isolated by FACS using established protocols to separate transit amplifying (TA), neuroblast (NB), activated stem cells (NSCs), and niche astrocytes (astros), and gene expression of each cell type was measured. All arrays are Nimblegen Mouse Gene Expression 12x135K Array.

长链非编码RNA（long noncoding RNAs, lncRNAs）已在细胞系及多种完整组织中被报道，但针对体内发育过程的长链非编码RNA分析仍较为有限。本研究针对成年小鼠室管膜下区（subventricular zone, SVZ）神经干细胞谱系的长链非编码RNA表达展开全面分析。本研究采用互补性全基因组技术手段，包括RNA测序（RNA-seq）、RNA捕获测序（RNA CaptureSeq）以及染色质免疫沉淀测序（ChIP-seq），将特定长链非编码RNA与神经细胞类型、发育过程及人类疾病状态建立关联。通过整合染色质状态图谱、定制化微阵列以及室管膜下区谱系的荧光激活细胞分选（fluorescence-activated cell sorting, FACS）数据，本研究严格筛选并鉴定出在成体神经发生中潜在发挥功能的长链非编码RNA。对其中两种长链非编码RNA——Six3os与Dlx1as——进行短发夹RNA（short hairpin RNA, shRNA）介导的敲低实验，结果表明长链非编码RNA在多能成体干细胞的胶质-神经元谱系特化过程中具有调控作用。综上，本研究的数据与分析流程为体内长链非编码RNA研究提供了一套极具连贯性的分析框架，同时为开发用于发育与疾病领域长链非编码RNA研究的易用型在线资源奠定了基础。本研究将室管膜下区单层培养细胞进行体外分化，分别培养1、2、4天后检测基因表达变化；同时采用已确立的实验方案，通过荧光激活细胞分选分离室管膜下区谱系细胞，分别获取转运扩增细胞（transit amplifying, TA）、成神经细胞（neuroblast, NB）、活化态神经干细胞（activated stem cells, NSCs）以及微环境星形胶质细胞（niche astrocytes, astros），并检测各类细胞的基因表达水平。本研究所用微阵列均为Nimblegen小鼠基因表达12×135K芯片。