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Series GSE59017 Query DataSets for GSE59017
Status Public on Jul 03, 2014
Title Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and dKO round spermatids Transcriptomes
Organism Mus musculus
Experiment type Expression profiling by high throughput sequencing
Summary 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.
Overall design Adult wild type (WT) and dKO mouse round spermatids were generated by deep sequencing, in dulplicate, using Illumina GAIIx.
Contributor(s) Smith RA, Tang C, Yu T, Yan W
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Submission date Jul 02, 2014
Last update date May 15, 2019
Contact name Wei Yan
Phone (775) 784-7765
Organization name University of Nevada
Department Physiology
Lab Wei Yan Lab
Street address 1664 N. Virginia Street
City reno
State/province NV
ZIP/Postal code 89557
Country USA
Platforms (1)
GPL11002 Illumina Genome Analyzer IIx (Mus musculus)
Samples (4)
GSM1424461 roundspd wt rep1
GSM1424462 roundspd wt rep2
GSM1424463 roundspd dKO rep1
BioProject PRJNA254123
SRA SRP044007

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Supplementary file Size Download File type/resource
GSE59017_gene_exp.diff.gz 1.4 Mb (ftp)(http) DIFF
GSE59017_isoform_exp.diff.gz 2.0 Mb (ftp)(http) DIFF
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Processed data are available on Series record
Raw data are available in SRA

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