Next Generation Sequencing for Quantitative Expression Analysis of Wild Type and Nrf1 deficient Liver Transcriptomes from Regular Diet (RD) or High Cholesterol Diet (CholD) fed mice

Purpose: Next-generation sequencing (NGS) for systematic analysis to discover Nrf1-dependent cellular pathways involved in defending liver against the stress of cholesterol excess. Methods: Liver mRNA profiles of 9-12-old wild-type (WT in fastq files; flox in paper) and liver Nrf1 deficient (KO in fastq files; L-KO in paper) mice were generated by deep sequencing, in triplicate or quadruplicate, using Illumina HiSeq2500. The sequence reads that passed quality filters were analyzed at the transcript level with TopHat followed by Cufflinks. qRT-PCR validation of selected target genes was performed using SYBR Green assays. Results: We mapped sequence reads to the mouse genome (build mm10) and identified greater than 10,000 transcripts in the liver of WT and KO mice. RNA-seq data revealed a role for Nrf1 in regulating inflammation, lipid metabolism, and sterol metabolism in response to a cholesterol challenge. Conclusion: Our study reveal Nrf1 as a guardian of cholesterol homeostasis and a core component of adaptive responses to excesses in cellular cholesterol. Liver mRNA profiles of 9-12 week old wild type (WT) and Nrf1 KO mice were generated by deep sequencing, in triplicate or quadruplicate, using Illumina HiSeq2500.研究目的：采用下一代测序（Next-generation sequencing, NGS）开展系统性分析，旨在发掘依赖于Nrf1的细胞通路，该通路参与保护肝脏抵御过量胆固醇引发的应激。 研究方法：针对9-12周龄野生型小鼠（fastq文件中记作WT，论文中记作flox）与肝脏Nrf1缺陷型小鼠（fastq文件中记作KO，论文中记作L-KO），通过Illumina HiSeq2500平台进行深度测序，测序重复次数为3次或4次。对通过质量过滤的序列读段，以转录本水平开展分析：先使用TopHat进行序列比对，再通过Cufflinks完成后续转录本分析。选取部分靶基因采用SYBR Green染料法进行实时定量逆转录PCR（quantitative reverse transcription PCR, qRT-PCR）验证。 研究结果：将序列读段比对至小鼠基因组（版本mm10），在野生型与缺陷型小鼠的肝脏中鉴定出超过10000个转录本。RNA测序数据显示，Nrf1在机体应对胆固醇刺激时，可调控炎症反应、脂质代谢与固醇代谢过程。 研究结论：本研究证实Nrf1是胆固醇稳态的守护因子，亦是细胞应对过量胆固醇的适应性应答核心组分。