Gene expression changes in dorsal dermal fibroblasts after beta-catenin deletion in vivo

Goal of this study was to examine gene expression changes upon conditional deletion of beta-catenin at E10.5 in dermal fibroblasts. Method: Skin tissues were treated in 0/25% trypsin for 15 min at 37 degrees C to obtain single cells for FACS sorting and collected in RNA later. RNA was extracted with Karcturus pico pure kit. The RT and amplification was done using Nugen's ovation RNA-seq system V2. Libraries for sequencing were prepared with Illumina TruSeq kit. Transcriptome of FACS sorted E13.5 Engrailed1; RRYFP lineage-marked dorsal dermal fibroblasts was generated next-gen sequencing, in triplicate, using Illumina HiSeq machine. The sequence reads that passed quality filters were analyzed by TopHat followed by Cufflinks. qRT-PCR validation was performed using TaqMan and SYBR Green assays Results: We mapped about 33-49 million sequence reads per sample and obtained 76-79% of uniquely mapped percentage. We assembled the reads to the mouse ref genome (build mm10). E13.5 dermal fibroblast mRNA profiles from En1;RRYFP (n=2) and En1Cre;RRYFP;beta-catenin flox/del (n=3) embryos (from three different litters) were generated by deep sequencing using Illumina Hi Seq本研究旨在探究在真皮成纤维细胞中于胚胎发育第10.5天（E10.5）条件性敲除β-连环蛋白（beta-catenin）后的基因表达变化。 实验方法：将皮肤组织置于37℃、0/25%胰蛋白酶中处理15分钟，以获取单细胞用于荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS），并将收集的细胞保存于RNA储存液（RNA later）中。采用Karcturus PicoPure试剂盒提取总RNA，使用Nugen公司Ovation RNA-seq系统V2完成反转录（Reverse Transcription, RT）与扩增。使用Illumina TruSeq试剂盒构建测序文库。随后，对经荧光激活细胞分选（FACS）获得的E13.5 Engrailed1;RRYFP谱系标记的背部真皮成纤维细胞进行转录组测序，设置三次生物学重复，采用Illumina HiSeq测序仪完成下一代测序（Next-Generation Sequencing, NGS）。对通过质量过滤的序列读段，先采用TopHat软件进行序列比对，再通过Cufflinks软件完成转录本分析。采用TaqMan与SYBR Green检测试剂盒完成定量实时聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）验证。 实验结果：每个样本可比对得到约3300万至4900万条序列读段，唯一比对率为76%至79%。我们将序列读段比对至小鼠参考基因组（版本mm10）。从3个不同胎仔中获取的En1;RRYFP（n=2）与En1Cre;RRYFP;beta-catenin flox/del（n=3）胚胎的E13.5真皮成纤维细胞mRNA表达谱，通过Illumina HiSeq深度测序获得。