Whole transcriptome profiling of Wildtype and EXT1-/- human embryonic stem cells by RNA-Seq

Purpose: The goals of this study are to identify whole transcriptomic changes between undifferentiated wildtype (H9 cell line) and undifferentiated EXT1-/- human embryonic stem cells using RNA-seq.Total RNA extraction was performed on low passage number wildtype and EXT1-/- human embryonic stem cells that were cultured in E8 media in quadruplicate. Libraries were prepared using TruSeq Stranded Total RNA kit (Illumina). First-strand cDNA was generated using random primers, followed by second-strand cDNA synthesis. Adapter-ligated PCR-enriched products were used to create cDNA libraries. 3' ends were adenylated and adapter ligated, followed by purification and enrichment with PCR to create cDNA libraries, which were sequenced using the Illumina platform. Single-end reads were mapped to the human genome using STAR. Mapped single-end reads were counted using RSEM.Results: We identified 1986 genes that were significantly differentially expressed [FDR (false discovery rate)-adjusted p < 0.05] between the two cell types. Of those genes, there were 102 upregulated and 195 downregulated transcripts that showed more than a two-fold change in expression. The top 20 upregulated and downregulated genes include those involved in neural development (e.g., POU3F4, ISLR2, FGF9), and transcription factors involved in early development (e.g., GRHL3, GBX2, ONECUT1). We evaluated the global changes in gene expression influenced by EXT1 deficiency compared with wildtype cells using Ingenuity Pathway Analysis. Gene ontology (GO) analysis of transcripts that significantly differ by more than two-fold revealed that the loss of EXT1 led to a significant enrichment for biological processes related to cell-to-cell signaling, development, and cellular functions.Conclusions: Our findings suggest that genes involved in human embryonic stem cell differentiation are differentially regulated in EXT1-/- cells and that therefore the capacity of the cells to differentiate may be compromised. Taken together, RNA-seq revealed key differences between wildtype and EXT1-/- cells at the transcript level.