Next Generation Sequencing Facilitates Quantitative Analysis of ribo-tag pulled down Transcriptomes in renal proximal tubule epithelial cells from Plasmodium Chabaudi Chabaudi infected Egfp.l10Pepck and Egfp.l10PepckSlc40a1Pepckdelta/delta mice. Next Generation Sequencing Facilitates Quantitative Analysis of ribo-tag pulled down Transcriptomes in renal proximal tubule epithelial cells from Plasmodium Chabaudi Chabaudi infected Egfp.l10Pepck and Egfp.l10PepckSlc40a1Pepckdelta/delta mice

Purpose: Next-generation sequencing (NGS) is emmerged as a highthrough-put technique to analyze cellular pathways. The goals of this study are to profile NGS-derived renal proximal tubule epithelial cells (RPTEC) transcriptome (RNA-seq), to identify transcription signature in RPTEC upon Plasmodium Chabaudi Chabaudi (Pcc) infection and steady state (NI). We also would like to understand how iron export affect the transcriptome in this cell compartment. Therefore, we will compare the transcriptome between Pcc infected (or steady state) Egfp.l10Pepck and Egfp.l10PepckSlc40a1Pepckdelta/delta mice. Methods: TRAP (Translating Ribosome Affinity Purification) were performed in the kidneys of Pcc infected (or steady state) 10-16 weeks Egfp.l10Pepck and Egfp.l10PepckSlc40a1Pepckdelta/delta mice. RPTEC TRAP mRNA profiles were generated by next generation sequencing, in triplicate, using Illumina High output kit v2.5 and NextSeq500 sequencer. Results: Using an optimized data analysis workflow, we mapped an average 32.54 million sequence reads per sample to the mouse genome (build mm39) and identified 19,052 transcripts in the TRAP mRNA of in the kidneys of Pcc infected or steady state 10-16 week old Egfp.l10Pepck and Egfp.l10PepckSlc40a1Pepckdelta/delta mice. Differential gene expression was performed using the DESeq2 R package (v.1.32) and gene expression was modeled by genotype and condition. Differentially expressed genes were considered for genes with an adjusted p-value0. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Overall design: TRAP (Translating Ribosome Affinity Purification) were performed in the kidneys of Pcc infected (or steady state) 10-16 weeks Egfp.l10Pepck and Egfp.l10PepckSlc40a1Pepckdelta/delta mice. RPTEC TRAP mRNA profiles were generated by next generation sequencing, in triplicate, using Illumina High output kit v2.5 and NextSeq500 sequencer.

研究目的：下一代测序（Next-generation sequencing, NGS）已成为分析细胞通路的高通量技术。本研究旨在对NGS来源的肾近端小管上皮细胞（renal proximal tubule epithelial cells, RPTEC）转录组（RNA-seq）进行谱型分析，以鉴定查氏疟原虫查氏亚种（Plasmodium Chabaudi Chabaudi, Pcc）感染及稳态（NI）下RPTEC的转录特征；同时拟阐明铁输出对该细胞亚群转录组的调控作用。为此，我们将比较Pcc感染（或稳态）的Egfp.l10Pepck与Egfp.l10PepckSlc40a1Pepckdelta/delta小鼠的转录组差异。

实验方法：对10-16周龄的Pcc感染（或稳态）Egfp.l10Pepck及Egfp.l10PepckSlc40a1Pepckdelta/delta小鼠的肾脏组织开展翻译核糖体亲和纯化（Translating Ribosome Affinity Purification, TRAP）实验。采用Illumina High output kit v2.5与NextSeq500测序仪，对RPTEC的TRAP mRNA进行高通量测序，每组设置三次生物学重复。

实验结果：通过优化的数据分析流程，我们将每个样本平均3254万条序列读数比对至小鼠基因组（版本mm39），并在Pcc感染或稳态的10-16周龄Egfp.l10Pepck及Egfp.l10PepckSlc40a1Pepckdelta/delta小鼠肾脏的TRAP mRNA中鉴定出19052个转录本。采用DESeq2 R包（v1.32）进行差异基因表达分析，以基因型与实验条件作为基因表达模型的协变量。筛选校正p值满足adjusted p-value0的基因作为差异表达基因。

研究结论：本研究首次通过RNA-seq技术完成了肾脏转录组的详细分析，且包含生物学重复样本。本研究报道的优化数据分析流程可为表达谱的比较研究提供分析框架。结果表明，NGS可实现细胞或组织内mRNA含量的全面、更精准的定量与定性评估。综上，基于RNA-seq的转录组表征可加速遗传网络分析，并助力解析复杂的生物学功能。

实验整体设计：对10-16周龄的Pcc感染（或稳态）Egfp.l10Pepck及Egfp.l10PepckSlc40a1Pepckdelta/delta小鼠的肾脏组织开展翻译核糖体亲和纯化（TRAP）实验。采用Illumina High output kit v2.5与NextSeq500测序仪，对RPTEC的TRAP mRNA进行高通量测序，每组设置三次生物学重复。