Integrative analysis of hepatic transcriptional profiles reveals genetic regulation of atherosclerosis in hyperlipidemic Diversity Outbred-F1 mice

Purpose: To investigate the sex-dependence of liver transcriptome in Diversity Outbred (DO)-F1 mice Methods: Total RNA was extracted from snap-frozen liver using miRVana total RNA isolation kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The quality and amount of liver RNA were evaluated using a Bioanalyzer (Agilent, Inc., Santa Clara, CA). The average RNA-integrity score for 162 DO-F1 liver samples was 9.01 ± 0.4. RNA samples from 85 females and 77 males were submitted to the UC Davis DNA Technologies Core at the Genome Center. The RNA-seq libraries were constructed from 1 µg total RNA after poly-A library preparation. To minimize technical variability, all samples were assigned to each lane and the pooled libraries were sequenced on two lanes of the Illumina NovaSeq 6000 sequencing (Illumina Inc., San Diego, CA, USA) to achieve paired-end reads of at least 25 million 150 bp. Only R1 was used in the analysis and only R1 was submitted. Results: Our results demonstrate the tremendous effects of sex on hepatic gene expression. In support of this, genetic loci associated with the transcripts frequently showed sex specificity. We revealed sex-specific candidate genes that were mapped to the quantitative trait loci for aortic lesion area and whose expression was regulated locally regulated via global liver transcriptome. Conclusions: Our study provide a valuable data resource to the research community and show that liver transcriptomic analysis identified diet- or strain-specific pathways to pathogenesis of metabolic syndrome. Overall design: Liver mRNA profiles of 24-week old Diversity Outbred-F1 mice研究目的：探究多样性远交（Diversity Outbred, DO）-F1小鼠肝脏转录组的性别依赖性。 实验方法：采用miRVana总RNA分离试剂盒（miRVana total RNA isolation kit，赛默飞世尔科技，美国马萨诸塞州沃尔瑟姆），按照制造商操作规程，从快速冷冻的肝脏组织中提取总RNA。使用生物分析仪（Bioanalyzer，美国加利福尼亚州圣克拉拉市安捷伦公司）评估肝脏RNA的质量与含量，162份DO-F1肝脏样本的RNA完整性评分平均值为9.01±0.4。将85份雌性样本与77份雄性样本的RNA提交至加州大学戴维斯分校基因组中心DNA技术核心实验室。以1μg总RNA为起始材料，经poly-A文库制备流程构建RNA测序文库。为最大限度降低技术偏差，所有样本均分配至各测序泳道，混合后的文库在Illumina NovaSeq 6000测序仪（Illumina Inc.，美国加利福尼亚州圣迭戈市Illumina公司）的两个泳道上进行测序，获得至少2500万条150bp的双端reads。本分析仅使用R1端测序数据，且仅提交了R1数据。 实验结果：本研究结果证实性别对肝脏基因表达具有显著影响。与此相符的是，与转录本相关的遗传位点常表现出性别特异性。本研究鉴定出性别特异性候选基因，这些基因被定位至主动脉病变面积相关的数量性状位点（quantitative trait loci），且其表达受肝脏全局转录组的局部调控。 研究结论：本研究为科研共同体提供了宝贵的数据资源，同时证实肝脏转录组分析可鉴定出与代谢综合征发病机制相关的饮食或品系特异性通路。 实验整体设计：24周龄多样性远交-F1小鼠的肝脏mRNA表达谱。