Transcriptomic analysis of C2C12 myotubes after adenoviral expression of Crebrf

Purpose: identify global changes in gene expression in C2C12 myotubes caused by an increase in Crebrf. Methods: Triplicate replicates of differentiated C2C12 myotubes expressing either Gfp or Crebrf for 3 days after transduction. After assessing RNA quality with Agilent Bioanalyzer, mRNAs were enriched by poly-A pull-down. Then, sequencing libraries constructed with Illumina TruSeq RNA prep kit were sequenced using. We multiplexed samples in each lane, which yields targeted number of single-end 75 bp reads for each sample, as a fraction of 180 million reads for the whole lane. Sequence reads were mapped back to the Drosophila genome (flybase genome annotation version r6.30) using STAR. With the uniquely mapped reads, we quantified gene expression levels using Cufflinks (FPKM values). Next, differentially expressed genes between experimental and control data were analyzed with DESeq2. Results: Gene set enrichment analysis reveals oxidative phosphorylation and interferon response as the top up- and down-regulated gene sets, respectively, upon Crebrf expression. The glycolysis gene set is also reduced. These gene expression findings parallel our metabolic observations in Seahorse analysis. Conclusions: Our study indicates that CREBRF is a strong regulator of muscle metabolism in C2C12 myotubes C2C12 myotubes were transduced by adenovirus encoding Gfp or Crebrf and RNA was isolated and analyzed by deep sequencing, in replicate, using Illumina HiSeq2000.

### 研究目的
鉴定Crebrf表达上调所引发的C2C12肌管（C2C12 myotubes）内基因表达的全局变化。

### 实验方法
设置生物学重复三组：将转导后培养3天的、分别表达绿色荧光蛋白（Gfp）与Crebrf的分化成熟C2C12肌管作为实验材料。首先使用安捷伦生物分析仪（Agilent Bioanalyzer）评估RNA质量，随后通过poly(A)下拉富集（poly-A pull-down）纯化mRNA。采用Illumina TruSeq RNA建库试剂盒（Illumina TruSeq RNA prep kit）构建测序文库并进行测序；本研究在每个测序泳道中开展多样本多重测序，将总泳道1.8亿条读段按比例分配至各样本，使每个样本获得预设数量的75 bp单端测序读段（reads）。测序读段通过STAR比对至果蝇基因组（flybase基因组注释版本r6.30）；利用唯一比对读段，采用Cufflinks计算FPKM值以定量基因表达水平，随后通过DESeq2分析实验组与对照组间的差异表达基因。

### 实验结果
基因集富集分析结果显示，在Crebrf表达后，氧化磷酸化通路为最显著上调的基因集，干扰素应答通路为最显著下调的基因集；糖酵解相关基因集的表达同样出现下调。上述基因表达特征与本研究通过Seahorse分析得到的代谢观测结果一致。

### 研究结论
本研究表明，CREBRF是分化成熟的C2C12肌管中肌肉代谢的关键调控因子。本研究通过腺病毒转染将编码Gfp或Crebrf的载体导入C2C12肌管，随后提取RNA并进行高通量测序，且设置了生物学重复，测序平台为Illumina HiSeq2000。