Determination of Sox10-regulated genes in the oligodendroglial precursor cell line OLN93

To determine Sox10-regulated genes in the oligodendroglial precursor cell line OLN93, CRISPR/Cas9-mediated genome editing was performed to inactivate Sox10. For CRISPR/Cas9-mediated Sox10 inactivation, two guide sequences (5’-GCTGCGAGCCAGCCCAGGGCC-3’ for clone A and 5’-GTTGCCCAATTCGCCG GGCCC-3’ for clone B) were separately cloned into pX330. Oln93 cells were co-transfected with pEGFP-N1 and the pX330 plasmids that express the respective guide RNA and SpCas9. Transfected GFP-positive cells were enriched by fluorescence activated cell-sorting and seeded at single cell density in 96-well plates. Homogeneously GFP-expressing clones were analysed for loss of Sox10 protein by immunocytochemistry. Clones without Sox10 protein were obtained with both guide RNAs. Sox10 inactivation was confirmed by Sanger sequencing of the Sox10 gene. RNA was isolated from one clone A and one clone B as well as two independent RNA samples of wildtype OLN93 cells and submitted to RNA-seq. mRNA profiles of control (ctrl) and genome-edited Sox10-deficient (Sox10ko) OLN93 cells were generated using Illumina HiSeq 2500 platform.

为鉴定少突胶质前体细胞系OLN93中Sox10调控的靶基因，本研究采用CRISPR/Cas9介导的基因组编辑技术灭活Sox10基因。针对CRISPR/Cas9介导的Sox10灭活，两条向导RNA序列（克隆A对应序列为5’-GCTGCGAGCCAGCCCAGGGCC-3’，克隆B对应序列为5’-GTTGCCCAATTCGCCGGGCCC-3’）被分别克隆至pX330载体中。将pEGFP-N1与表达对应向导RNA和SpCas9的pX330质粒共转染OLN93细胞，通过荧光激活细胞分选术富集GFP阳性细胞，并以单细胞密度接种于96孔培养板中。对均匀表达GFP的克隆开展免疫细胞化学检测，以分析Sox10蛋白的缺失情况；两条向导序列均成功获得了Sox10蛋白缺失的克隆。Sox10的基因灭活效果通过Sanger测序得到验证。分别从1株克隆A、1株克隆B以及两批独立的野生型OLN93细胞中提取总RNA，提交进行RNA测序（RNA-seq）。采用Illumina HiSeq 2500平台获取了对照组（ctrl）与Sox10缺陷型（Sox10ko）OLN93细胞的mRNA表达谱。