Next generation sequencing analysis of DN1 transcriptomes from Wild type, gl/gl and transgenic CD2-Ostm1 gl/gl mice

The goal of this study is to compare the transcriptome stability of DN1wt versus DN1gl/gl and DN1CD2-Ostm1gl/glTR Methods: DN1 thymocytes mRNA profiles of 19-day-old wild-type (WT), osteopetrotic grey-lethal (gl/gl) and transgenic CD2-Ostm1 gl/gl mice were generated by deep sequencing,DN1 cells from 2-3 mice per genotype were pooled, no technical replicates, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: TopHat and Cufflinks. qRT-PCR validation was performed using SYBR Green assays Results: DN1 gl/gl showed a distinctive transcriptome signature in comparison to DN1 wt and DN1 gl/glTR with respectivelly 146 and 205 differentially expressed genes and 205. Migration genes were significantly affected in DN1 gl/gl samples and RAC1 and S1PR1 gene expressions were confirmed using RT-qPCR. Conclusions: RNA seq allowed us to identify the enhanced expression of migration genes (RAC1 and S1PR1) in DN1gl/gl, that were both normalized in the DN1 cells from transgenic gl/glTR mice, which suggests defective T cell migration associated to the osteopetrotic thymus phenotype. DN1 thymocytes mRNA profiles of 19-day-old wild-type (WT), osteopetrotic grey-lethal (gl/gl) and transgenic CD2-Ostm1 gl/gl mice were compared by deep sequencing,DN1 cells from 2-3 mice per genotype were pooled, no technical replicates, using Illumina HiSeq 2000.本研究旨在比较DN1野生型（DN1wt）、DN1gl/gl以及DN1CD2-Ostm1gl/glTR的转录组（transcriptome）稳定性。方法：本研究通过深度测序（deep sequencing）获取19日龄野生型（WT）、骨质硬化灰色致死（gl/gl）以及转基因CD2-Ostm1 gl/gl小鼠的DN1胸腺细胞mRNA表达谱。每个基因型取2-3只小鼠的DN1细胞混合，未设置技术重复，测序平台为Illumina HiSeq 2000。对通过质量过滤的序列读段，采用TopHat和Cufflinks两种方法在转录本异构体层面进行分析。采用SYBR Green分析法进行实时定量逆转录聚合酶链反应（quantitative real-time polymerase chain reaction，qRT-PCR）验证。结果：相较于DN1野生型与DN1 gl/glTR，DN1 gl/gl呈现独特的转录组特征，分别存在146和205个差异表达基因。迁移相关基因在DN1 gl/gl样本中显著受影响，且RAC1与S1PR1基因的表达经RT-qPCR得到验证。结论：RNA测序（RNA-seq）使我们得以鉴定出DN1gl/gl中迁移相关基因（RAC1与S1PR1）的表达上调，而这些基因在转基因gl/glTR小鼠的DN1细胞中均恢复至正常水平，这表明与骨质硬化性胸腺表型相关的T细胞迁移存在缺陷。