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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE144025 | GEO | 2020/01/22

REPOSITORIES: GEO

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Project description:We determined the genome-wide digital gene expression (DGE) profiles of primary acute lymphoblastic leukemia (ALL) cells from 28 patients and fractionated blood cells from healthy blood donors taking advantage of “second generation” sequencing technology. The patients included in the study represent distinct subtypes of B-cell precursor (BCP) ALL and T-cell lineage ALL (T-ALL) and the controls are fractionated CD19+ and CD3+ cells. Gene expression analysis of 28 ALL patient samples with different immunophenotypic backgrounds including T-ALL (n=4) and patients with BCP ALL with diverse cytogenetic backgrounds: High Hyperploidy (HeH) (n=10), t(9;22) BCR-ABL1 (n=3), t(12;21) ETV6-RUNX1 (n=4), dic(9;20) (n=3), t(1;19)TCF3-PBX1, MLL/11q23 (n=1) and undefined/non-recurrent aberrations (n=1). Fractionated b-cells (CD19+) and t-cells (CD3+) isolated from peripheral blood of healthy donors were used as controls. Sequencing libraries were prepared from 1 µg of total RNA using reagents from the NlaIII Digital Gene Expression Tag Profiling kit (Illumina Inc, San Diego, CA, USA). mRNA was captured on magnetic oligo(dT) beads and reverse transcribed into double-stranded cDNA. The cDNA was cleaved using the restriction enzyme NlaIII. An adapter sequence containing the recognition sequence for the restriction enzyme MmeI was ligated to the NlaIII cleavage sites. The adapter-ligated cDNA was digested with MmeI to release the cDNA from the magnetic bead, while leaving 17 base-pairs of sequence in the fragment. The fragments were dephosphorylated and purified by phenol-chloroform. A second adapter was ligated at the MmeI cleavage sites. The adapter-ligated cDNA fragments were amplified by PCR, and the PCR products were purified on a 6% polyacrylamide gel. The ~96 base pair PCR products were excised from the gel and eluted overnight, followed by ethanol precipitation and re-suspension. Purified libraries were quality controlled and quantified on a Bioanalyzer using DNA 1000 series or High Sensitivity chips. The DGE libraries were diluted to a 10 nM concentration and sequenced on one lane of an Illumina GAII or GAIIx for 18 cycles.

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

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