Transcriptome profiling of wild type primordial germ cells (PGC) by RNAseq

Purpose: To identify molecular pathways underlying epigenetic reprogramming in early germ cell precursors, we examined global gene expression of wild type primordial germ cells using mRNA sequencing. Methods: Given the limited number of PGCs collected from E9.5 to E13.5 (ranging from 300 to 5000), we used a low-input RNA sequencing method, Smart-Seq. RNA libraries were pooled and sequenced by Illumina Hiseq. Results: We generated >22 million uniquely mapped reads per sample and identified >10 thousand transcripts per genotype (RPKM>0.1). Hierarchical clustering and correlation analysis on gene expression indicates samples were clearly separated according to their genotypes with Spearman correlation coefficient of 0.98/0.99 within biological replicates. Compared with E9.5 PGCs, 479 genes were significantly up-regulated and 248 genes were down-regulated in E11.5 PGCs. When compared with E11.5 PGCs, male E13.5 PGCs had 362 up-regulated, and 239 down-regulated genes, whereas female E13.5 PGCs had 1163 up-regulated and 333 down-regulated genes. Overall, the number of up-regulated genes was greater than that of the down-regulated genes in every comparison, suggesting that gene expression is generally activated during PGC reprogramming. mRNA profiles of primordial germ cells derived from developmental embryos stages (E9.5, E11.5 and E13.5) were generated by deep sequencing, in duplicates (E9.5 and E11.5) or triplicates (E13.5f and E13.5m), using Illumina Hiseq.研究目的：为明确早期生殖细胞前体表观遗传重编程的分子通路，我们采用mRNA测序（mRNA sequencing）技术分析野生型原始生殖细胞（primordial germ cells，PGCs）的全局基因表达谱。实验方法：鉴于从E9.5至E13.5阶段采集的PGCs数量有限（300至5000个不等），我们使用了低起始量RNA测序方法Smart-Seq。将构建完成的RNA文库混合后，通过Illumina Hiseq平台完成测序。实验结果：本研究每个样本可生成超过2200万条唯一比对读数，每个基因型样本可鉴定出超过1万条转录本（RPKM>0.1）。对基因表达数据进行层次聚类（hierarchical clustering）与相关性分析后发现，样本可依据基因型清晰分离，生物学重复样本间的斯皮尔曼相关系数（Spearman correlation coefficient）可达0.98/0.99。与E9.5阶段的PGCs相比，E11.5阶段的PGCs中有479个基因显著上调、248个基因显著下调。与E11.5阶段的PGCs相比，雄性E13.5阶段的PGCs存在362个上调基因与239个下调基因，而雌性E13.5阶段的PGCs则有1163个上调基因与333个下调基因。整体而言，所有比较组中上调基因的数量均多于下调基因，提示在PGCs重编程过程中基因表达整体处于激活状态。本研究通过Illumina Hiseq平台开展深度测序，获取了不同发育阶段（E9.5、E11.5及E13.5）胚胎来源的原始生殖细胞的mRNA表达谱，其中E9.5与E11.5组设置2次生物学重复，E13.5雌性组与E13.5雄性组设置3次生物学重复。