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Transcriptome profiling of Ccng1-/-Ccng2-/- double knockout (DKO) deficient mouse embryonic fibroblasts

ABSTRACT: To examine if lack of CycG1 and/or CycG2 causes genome-wide alterations of gene expression, we performed DNA microarray analysis on G1KO, G2KO and DKO MEFs at their logarithmic growth phase. Notably, the fold change values are much larger in DKO MEFs compared with those of G1KO or G2KO MEFs, which suggested that loss of both CycG1 and CycG2 caused uncontrolled expression of these genes because they are mutually regulated by CycG1 and CycG2. Moreover, the fold change of up- or down-regulated genes in DKO MEF are much larger than those of G1KO or G2KO MEFs, suggesting that functions of CycG1 and CycG2 somehow overlap and complement each other in G1KO and G2KO MEFs.

ORGANISM(S): Mus musculus

PROVIDER: GSE79617 | GEO | 2016/12/19

SECONDARY ACCESSION(S): PRJNA316454

REPOSITORIES: GEO

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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived WT and dKO round spermatids transcriptome profiling (RNA-seq) Methods: Adult WT and dKO round spermatids mRNA profiles mice were generated by deep sequencing, in dulplicate. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl−/− mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and dKO round spermatids, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, 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. Adult wild type (WT) and dKO mouse round spermatids were generated by deep sequencing, in dulplicate, using Illumina GAIIx.

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Transcriptome profiling of Ccng1-/-Ccng2-/- double knockout (DKO) deficient mouse embryonic fibroblasts

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