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Next-generation Sequencing Facilitates Quantitative Analysis of Wild Type and EED cKO mice Hippocampus Dentate Gyrus Transcriptomes

ABSTRACT: Purpose:Quantification differentially expressed genes and find target genes. The goals of this study are to compare Wild Type and EED cKO hippocampus dentate gyrus transcriptome profiling (RNA-seq) to quantitative reverse transcription polymerase chain reaction (qRT–PCR) differentially expressed genes and find target genes Methods: Hippocampus dentate gyrus mRNA profiles of 14-day-old wild-type (WT) and Embryonic Ectoderm Development conditional knockout (EED cKO) mice were generated by deep sequencing, in two biological replicates,using BGISEQ-500 platform.All RNA-Seq data were aligned to mouse genome version mm10 using HISAT.We use cluster software and Euclidean distance matrix for the hierarchical clustering analysis of the expressed gene and sample program at the same time, the clustering results can be viewed with javaTreeview. qRT–PCR validation was performed using SYBR Green assays. Results:Only transcripts that showed more than 2-fold differential expression compared to control were subjected to relevance network analysis. 1047 candidate genes that were differentially expressed with biological functional groups as compared between WT and EED cKO mice . Conclusions: Our study represents the first detailed analysis of Wild Type and EED cKO mice hippocampus dentate gyrus transcriptomes, with two biologic replicates, generated by RNA-seq technology.

ORGANISM(S): Mus musculus

PROVIDER: GSE131035 | GEO | 2019/05/11

REPOSITORIES: GEO

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Project description:Neural stem cells divide throughout adulthood in neurogenic niches - the dentate gyrus of the hippocampus and the subventricular zone (SVZ) - producing progenitor cells and new neurons. Moreover, stem cells self-renew, thus preserving their pool. The number of stem/progenitor cells in the neurogenic niches declines with age. We have previously demonstrated that the cyclin-dependent kinase inhibitor p16Ink4a maintains, in aged mice, the pool of dentate gyrus stem cells by preventing their activation after a neurogenic stimulus such as exercise (running). We showed that, although p16Ink4a ablation by itself does not activate stem/progenitor cells, exercise strongly induced stem cell proliferation in p16Ink4a knockout dentate gyrus, but not in wild-type. As p16Ink4a regulates stem cell self-renewal during aging, we sought to profile the dentate gyrus transcriptome from p16Ink4a wild-type and knockout aged mice, either sedentary or running for 12 days. By pairwise comparisons of differentially expressed genes and by correlative analyses through the DESeq2 software, we identified genes regulated by p16Ink4a deletion, either without stimulus (running) added, or following running. The p16Ink4a knockout basic gene signature, i.e. in sedentary mice, involves up-regulation of apoptotic, neuroinflammation- and synaptic activity-associated genes, suggesting a reactive cellular state. Conversely, another set of 106 genes we identified, whose differential expression specifically reflects the pattern of proliferative response of p16 knockout stem cells to running, are involved in processes regulating stem cells activation, i.e., synaptic function, neurotransmitter metabolism, stem cell proliferation control, and regulation of ROS levels. Moreover, we analyzed the regulation of these stem cell-specific genes after a second running stimulus. Surprisingly, the second running neither activated stem cell proliferation in the p16Ink4a knockout dentate gyrus nor changed the expression of these genes, confirming that they are correlated to the stem cell reactivity to stimulus.

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Next-generation Sequencing Facilitates Quantitative Analysis of Wild Type and EED cKO mice Hippocampus Dentate Gyrus Transcriptomes

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