Quantitative Analysis of Follicular Helper T cell Transcriptomes from Wild Type and Apoe-/-. Quantitative Analysis of Follicular Helper T cell Transcriptomes from Wild Type and Apoe-/-

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goal of this study is to characterize the pathogenic features of follicular helper T cells (TFH cells) generated in atherogenic conditions by NGS-derived transcriptome profiling (RNA-seq). Methods: TFH cell mRNA profiles of 13-week-old wild type (WT) and Apoe knockout (Apoe-/-) mice were generated by deep sequencing, in duplicate, using Invitrogen mirVana miRNA Isolation Kit (Cat#AM1561). The sequence reads that passed quality filters were analyzed by TopHat (version 2.0.13) followed by Cufflinks (version 2.2.0). Results: Using an optimized data analysis workflow, we mapped about 50 million sequence reads per sample to the mouse genome (build mm10) and identified 23997 transcripts in the retinas of WT and Apoe–/– mice with approximately 88% mapping rates. At a setting of greater than two-fold expression changes, p value < 0.05, and false discovery rate (FDR) < 0.1, 211 genes were upregulated and 142 genes were downregulated in TFH cells from Apoe–/–mice compared with those from WT mice. Conclusions: Our study demonstrates detailed transcriptome analysis of TFH cells from WT and Apoe–/–, with biologic replicates, generated by RNA-seq technology. Overall design: mRNA profiles of follicular T helper cells from wild type and atherogenic mice were generated by deep sequencing, in duplicate, using Illumina Truseq.

研究目的：下一代测序（NGS）已革新了细胞通路的系统生物学分析范式。本研究旨在通过NGS衍生的转录组测序（RNA-seq）技术，对致动脉粥样硬化条件下诱导产生的滤泡辅助性T细胞（TFH细胞）的致病特征进行系统表征。
研究方法：采用Invitrogen mirVana miRNA分离试剂盒（货号AM1561），对13周龄野生型（WT）及载脂蛋白E敲除（Apoe-/-）小鼠的TFH细胞进行深度测序，每份样本设置双生物学重复以获取其mRNA表达谱。对通过质量过滤的测序读段，先使用TopHat（版本2.0.13）进行基因组比对，随后采用Cufflinks（版本2.2.0）进行转录组分析。
研究结果：借助优化后的数据分析流程，我们将每个样本约5000万条测序读段比对至小鼠基因组（版本mm10），在野生型及Apoe-/-小鼠的视网膜组织中鉴定出23997个转录本，整体比对率约为88%。在表达差异倍数≥2、p值<0.05且错误发现率（FDR）<0.1的筛选标准下，相较于野生型小鼠，Apoe-/-小鼠的TFH细胞中共有211个基因上调、142个基因下调。
研究结论：本研究借助RNA-seq技术并设置生物学重复，完成了野生型及Apoe-/-小鼠TFH细胞的详尽转录组分析。
实验整体设计：采用Illumina Truseq建库体系，通过深度测序（每份样本设置双生物学重复）获取野生型小鼠与致动脉粥样硬化模型小鼠的滤泡辅助性T细胞mRNA表达谱。