RNA-Seq on libraries made from serial dilutions of Drosophila melanogaster S2 cell mRNA and ERCC external RNA controls

RNA-Seq on libraries made from serial dilutions of mRNA from Drosophila melanogaster S2 cell and the External RNA Controls Consortium (ERCC) external RNA controls. We evaluated performance of RNA-Seq by serially diluting a complex pool of known synthetic PolyA+ mRNAs from the External RNA Controls Consortium (ERCC) and PolyA+ mRNA from Drosophila S2 cells. ERCC mRNAs were obtained under Phase V testing from the National Institutes of Standards and Technology (NIST). The ERCC pool contained 96 species of mRNA of various lengths and GC content covering a 2^20 concentration range. Libraries were constructed with 100ng to 10pg of input mRNA. Our data shows an outstanding linear fit between RNA-Seq read density and known input amounts. We performed RNA-Seq from libraries made with 0.01ng to 100ng mRNA (mixture of mRNA from Drosophila melanogaster S2 cells and ERCC RNA controls). One RNA-Seq library was prepared with 100ng mRNA and six libraries were made with serial dilutions of mRNA using a modified protocol (see Sample 'extraction protocol'). One lane was sequenced for each library and all seven libraries were run on the same flow cell.

本数据集针对由黑腹果蝇（Drosophila melanogaster）S2细胞mRNA与外部RNA对照联盟（External RNA Controls Consortium, ERCC）外部RNA对照品经系列稀释后构建的测序文库开展RNA测序（RNA-Seq）。本研究通过对外部RNA对照联盟（ERCC）的已知合成聚腺苷酸化（PolyA+）mRNA混合池以及黑腹果蝇S2细胞的PolyA+ mRNA进行系列稀释，评估了RNA-Seq的检测性能。本次研究所用的ERCC mRNA购自美国国家标准与技术研究院（National Institutes of Standards and Technology, NIST）的V期测试项目。该ERCC混合池包含96种不同长度、不同GC含量的mRNA，其浓度覆盖范围达2^20量级。测序文库的构建起始mRNA投入量为100ng至10pg。本研究数据显示，RNA-Seq的读段密度与已知的mRNA投入量之间存在极佳的线性拟合关系。我们对由0.01ng至100ng的mRNA（黑腹果蝇S2细胞mRNA与ERCC RNA对照品的混合体系）构建的文库开展了RNA-Seq测序。其中1个文库采用100ng mRNA构建，另有6个文库通过改良实验方案（详见样本‘提取实验方案’）以系列稀释的mRNA构建而成。每个文库均采用单个泳道进行测序，且全部7个文库均在同一流动槽（flow cell）上完成测序。