Next Generation Sequencing Facilitates Quantitative Analysis of Cyp26b1-/-skin and En1cre;Cyp26b1f/- epidermal and dermal Transcriptomes

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived skin transcriptome profiling (RNA-seq) to determine pathways and networks dependent on retinoic acid during skin development. Methods: Skin mRNA profiles of embryonic day E16.5 wild-type (WT) and Cyp26b1 knockout (Cyp26b1-/-), and of control and of dermal and epidermal skin fractions of Engrailed1cre;Cyp26b1f/- (En1cre;Cyp26b1f/-) conditional knockout mice were generated by deep sequencing, in duplicate, using Illumina HiSeq2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level by ANOVA (ANOVA) and TopHat. qRT-PCR validation was performed using TaqMan and SYBR Green assay. Results: RNA-Seq data were generated with Illumina's HiSeq 2000 system. Raw sequencing data were processed with CASAVA 1.8.2 to generate fastq files. Reads of 50 bases were mapped to the mouse transcriptome and genome mm9 using TopHat 1.3.2. Gene expression values (RPKM) were calculated with Partek Genomics Suite 6.6, which was also used for the ANOVA analysis to determine significantly differentially expressed genes. Conclusions: Our study represents the first detailed analysis of Cyp26b1-/- skin and En1cre;Cyp26b1f/- dermis/epidermic 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. 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 embryonic-day 16.5 wild type (WT) and Cyp26b1-/- mice and of dermis and epidermis of embryonic day 18.5 control and En1cre;Cyp26b1f/- were generated by deep sequencing, in duplicate, using Illumina HiSeq2000.研究目的：下一代测序（Next-generation sequencing, NGS）已彻底革新了基于系统生物学的细胞通路分析方法。本研究旨在对比基于NGS获得的皮肤转录组测序数据（RNA-seq），以解析皮肤发育过程中依赖视黄酸的信号通路与调控网络。 方法：本研究采用Illumina HiSeq2000平台进行双份深度测序，分别获取了胚胎第16.5天（E16.5）野生型（wild-type, WT）与Cyp26b1基因敲除（Cyp26b1-/-）小鼠的皮肤mRNA表达谱，以及Engrailed1cre;Cyp26b1f/-（En1cre;Cyp26b1f/-）条件性敲除小鼠的真皮、表皮组分的皮肤mRNA表达谱。通过质量过滤的序列读数将在转录本异构体水平上通过方差分析（ANOVA）与TopHat工具进行分析。采用TaqMan探针法与SYBR Green染料法进行qRT-PCR验证。 结果：本研究通过Illumina HiSeq 2000系统生成了RNA-seq数据。原始测序数据经CASAVA 1.8.2处理以生成fastq格式文件。将50碱基长度的测序读数比对至小鼠转录组与mm9版本参考基因组，比对工具为TopHat 1.3.2。基因表达值（每百万映射reads每千碱基转录本片段数，RPKM）通过Partek Genomics Suite 6.6计算得到，该工具同时用于方差分析以筛选显著差异表达基因。 结论：本研究首次对Cyp26b1-/-小鼠皮肤以及En1cre;Cyp26b1f/-小鼠的真皮/表皮转录组进行了详细分析，所有样本均设置生物学重复，测序技术采用RNA-seq。本文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框架。我们认为，基于RNA-seq的转录组表征可加速遗传网络分析，并为解析复杂生物学功能提供可能。本研究通过Illumina HiSeq2000平台进行双份深度测序，获取了胚胎第16.5天野生型与Cyp26b1-/-小鼠的皮肤mRNA表达谱，以及胚胎第18.5天对照组与En1cre;Cyp26b1f/-小鼠的真皮、表皮组分的皮肤mRNA表达谱。