Next Generation Sequencing Facilitates Quantitative Analysis of WT MRSA USA300 Infected HEKn and HaCaT Transcriptomes

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Method: HaCaTs and primary keratinocytes were infected with WT USA300 S. aureus (MOI 100:1) for 1 hour before cell lysates were prepared. Poly-A pull-down was used to enrich mRNAs from total RNA samples (200ng-1ug per sample, RIN>8 required) and to proceed to library preparation by using Illumina TruSeq RNA prep kit. Libraries were then sequenced using Illumina HiSeq2500 at Columbia Genome Center. The samples were multiplexed in each lane, which yields targeted number of singleend/paired-end 100bp reads for each sample, as a fraction of 180 million reads for the whole lane. The raw read files were not available for submission because they have been deleted by the core facility. Results: Using an optimized data analysis workflow, we identified 15,780 transcripts in the skin of both HEKn and HaCaT cells. Expression values were analyzed using Ingenuity Pathway Analysis (IPA). Conclusions: Our study represents the first detailed analysis of HEKn and HaCaT transcriptomes, with biologic replicates, generated by RNA-seq technology after WT MRSA USA300 infection. 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. Skin mRNA profiles of HEKn and HaCaT cells were generated by deep sequencing, in triplicate, using Illumina HiSeq2500

### 研究目的
下一代测序（Next-generation sequencing, NGS）已彻底革新了基于系统视角的细胞通路分析。本研究旨在对比NGS衍生的视网膜转录组测序（RNA-seq）与微阵列、定量逆转录聚合酶链反应（quantitative reverse transcription polymerase chain reaction, qRT–PCR）的检测性能，并评估用于最优高通量数据分析的实验方案。

### 实验方法
将HaCaT细胞与原代角质形成细胞以野生型USA300金黄色葡萄球菌（Staphylococcus aureus, S. aureus）按感染复数（multiplicity of infection, MOI）100:1的比例感染1小时，随后制备细胞裂解液。采用Poly-A下拉法从总RNA样本（每份样本200ng~1μg，要求RNA完整性指数RIN>8）中富集mRNA，并使用Illumina TruSeq RNA建库试剂盒完成文库构建。将构建好的文库送至哥伦比亚基因组中心，采用Illumina HiSeq2500测序平台进行测序。每个测序泳道均采用多样本混样策略，每个样本可获得目标数量的单端/双端100bp读段，占单泳道总读段数（1.8亿）的一定比例。由于核心测序设施已删除原始读段文件，故无法提交原始测序数据。

### 研究结果
通过优化的数据分析流程，我们在HEKn与HaCaT细胞中共计鉴定出15780条转录本。采用Ingenuity Pathway Analysis（IPA）对基因表达量数据进行分析。

### 研究结论
本研究首次针对野生型耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus, MRSA）USA300感染后的HEKn与HaCaT细胞，开展了带有生物学重复的RNA-seq转录组详细分析。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标准化分析框架。结果表明，NGS可对细胞或组织内的mRNA含量实现全面且更为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。本研究通过Illumina HiSeq2500平台完成了HEKn与HaCaT细胞的皮肤mRNA深度测序，每个样本设置3次生物学重复。