GSE118542
RNA-seq Quantitative Analysis of Wild Type and Kcnh2-/- day4 cells derived from rESCs
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. To understand how ERG1 executes its function during differentiation, we performed RNA-seq experiments in Kcnh2-/- and wild-type rEBs at day 4 Methods: RNA profiles of day 4  wild type (WT) and Kcnh2-/- rEBs were generated by deep sequencing, in duplicate, using Illumina Hiseq. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks. Results: Using an optimized data analysis workflow, we mapped about 35 million sequence reads per sample to the rat genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Kcnh2-/- rESC  with TopHat workflow. RNA-seq datashows 211 differentially expressed gene with a fold change =2 and p value <0.01, in which 109 genes were downregulated and 102 genes were upregulateds.Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method.Based on GO enrichment analysis results, the deletion of Kcnh2 resulted in the impaired heart development and cell differentiation by inhibiting the expression of heart related genes. KEGG enrichment analysis of all differentially expressed genes yielded the enrichment for several cell differentiation signaling pathways, like PI3K-Akt signaling pathway and Wnt signaling pathway, which is  consistent with the experiment results. Conclusions: Our study represents the detailed analysis of transcriptomes of Kcnh2-/- vs. WT  rat embryonic stem cell, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Overall design: MRNA profiles of  day 4  wild type (WT) and Kcnh2-/- rEBs  were generated by deep sequencing, in duplicate, using Illumina Hiseq.