RNA-seq of SOX5 overexpressing primary human neuronal progenitors

Purpose: The goal of this study was to assess gene expression changes in neurons overexpressing SOX5 using human primary neuronal culture system. Methods: 6 samples each from control GFP and SOX5 overexpressing neurons were used to isolate total RNA using miRNeasy kit, Qiagen. We performed rRNA-depleted 69bp paired end stranded RNA-seq on neurons overexpressing either GFP or SOX5 tagged with GFP. Overexpression of SOX5 in neurons validated that a significant proportion of Attenuated cortical patterning (ACP) genes are regulated by SOX5, and that predicted SOX5 targets exhibit a net downregulation, consist with its repressive function. This supports the prediction that attenuated patterning of SOX5 between cortical regions contributes to direct alterations in SOX5 targets and likely to indirect alterations in SOX5 non-targets in the ACP set. delpleted 69 bp stranded RNA-seq in SOX5 was overexpressed in primary human neuronal cultures using a lentiviral system. Briefly full-length human SOX5 gene was cloned in pLVU/GFP vector (gift from Lars Ittner [Addgene plasmid #24177]) using the gateway recombination technique. Lentivirus was produced in HEK293T cells using a second generation packaging vector system (psPAX2, a gift from Didier Trono [Addgene plasmid #12260] and pCMV-VSV-G, a gift from Bob Weinberg [Addgene plasmid #8454]) as described by Stewart et al., 200331. Primary human neurons were infected at plating at a multiplicity of infection (MOI) of 10 with either the SOX5 overexpressing construct or a control pLVU-GFP backbone vector. 14 days after infection, RNA from the samples were isolated using miRNeasy micro kit (Qiagen, Carlsbad) and 50bp paired-end libraries were prepared using SMARter Stranded Total RNA sample prep kit (Clontech) with rRNA depletion. Libraries were then multiplexed and sequenced with HiSeq 2500 instrument (Illumina).

研究目的：本研究旨在借助人类原代神经元培养体系，评估过表达SOX5的神经元内的基因表达变化情况。
实验方法：分别从转染对照GFP的神经元与过表达GFP标签化SOX5的神经元中各获取6份样本，使用Qiagen公司的miRNeasy试剂盒提取总RNA。我们对两组神经元样本开展了rRNA去除的69bp双端链特异性RNA测序。
在神经元中过表达SOX5后，我们验证发现，相当比例的皮质减损模式（Attenuated cortical patterning, ACP）基因受SOX5调控，且预测的SOX5靶基因整体呈现下调趋势，这与SOX5的转录抑制功能相一致。该结果支持以下推测：大脑皮层区域间SOX5的减损表达模式，会直接改变SOX5靶基因的表达，并可能间接调控ACP基因集中非SOX5靶基因的表达。
本研究通过慢病毒载体系统在原代人神经元中过表达SOX5，并开展rRNA去除的69bp链特异性RNA测序。简言之，我们通过Gateway重组技术将全长人SOX5基因克隆至pLVU/GFP载体（由Lars Ittner惠赠，Addgene质粒#24177）。按照Stewart等人2003年的方法，使用第二代包装载体系统（psPAX2，由Didier Trono惠赠，Addgene质粒#12260；以及pCMV-VSV-G，由Bob Weinberg惠赠，Addgene质粒#8454）在HEK293T细胞中包装慢病毒。原代人神经元在接种时以感染复数（multiplicity of infection, MOI）10分别感染SOX5过表达构建体或对照pLVU-GFP空载体。感染14天后，使用miRNeasy微试剂盒（Qiagen，卡尔斯巴德）提取样本总RNA，并使用SMARter链特异性总RNA样本制备试剂盒（Clontech）进行rRNA去除，制备50bp双端测序文库。随后将文库进行多重复合标记，使用HiSeq 2500测序仪（Illumina）完成测序。