Profiling of Stem/Progenitor Cell Regulatory Genes of the Synovial Joint by Genome-wide RNASeq Analysis

We report the application of RNA sequencing technology for high-throughput profiling of the articular and mature zones of the mandibular condyle in postnatal 7-day-old mice. Briefly, mandibular condyles of postnatal 7-day-old mice were frozen-sectioned with articular, mature zones identified under a dissection microscope. Cells from the articular zone (az), mature zone (mz) were isolated by laser capture microscopy. Total RNAs from each sample were polyA selected and single-end sequencing libraries were constructed using TruSeq RNA Sample Prep Kit (Illumina). The samples were then sequenced using the Illumina HiSeq sequencer. Total sequencing reads representing RNA transcripts were generated and were mapped to the NCBI mouse reference genome. Differential expressed genes were identified using DESeq, with <0.05 false discovery rate (FDR). Fragments Per Kilobase of exon model per Million Mapped fragments (FPKM) were estimated for each gene in each sample. Hierarchical clustering was also performed on log2FPKM values with Cluster 3.0. Only genes with FDR<0.05 and >=0.80 standard deviation were included. RNA comparison between articular and mature zones revealed distinctive gene expression profiles. Profiling of the articular and mature zones of postnatal 7-day-old mice mandibular condyle by RNA sequencing, in triplicate, using Illumina HiSeq sequencer.本研究报道了RNA测序技术在出生后7日龄小鼠下颌髁突关节区与成熟区高通量转录组谱分析中的应用。简要而言，我们对出生后7日龄小鼠的下颌髁突进行冰冻切片，并在解剖显微镜下区分关节区与成熟区；通过激光捕获显微切割技术分离得到关节区（az）与成熟区（mz）的细胞。提取各样本的总RNA后进行polyA富集，并使用Illumina公司的TruSeq RNA样本制备试剂盒构建单端测序文库，随后采用Illumina HiSeq测序仪完成测序。本研究产出了代表RNA转录本的总测序读数，并将其比对至NCBI小鼠参考基因组。使用DESeq软件鉴定差异表达基因，筛选阈值为错误发现率（false discovery rate, FDR）<0.05；为每个样本中的每个基因估算每百万比对片段的外显子模型每千碱基片段数（Fragments Per Kilobase of exon model per Million Mapped fragments, FPKM）。使用Cluster 3.0软件对log₂转化后的FPKM值进行层级聚类分析，仅纳入FDR<0.05且标准差≥0.80的基因。关节区与成熟区的RNA转录本比较结果显示二者具有显著差异的基因表达谱。本研究采用Illumina HiSeq测序仪，对出生后7日龄小鼠下颌髁突的关节区与成熟区进行了三次生物学重复的RNA测序谱分析。