RNA-Seq and RNA Polymerase II ChIP-Seq of mouse spermatogenesis
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE44346 | GEO | 2014/01/22
SECONDARY ACCESSION(S): PRJNA189510
REPOSITORIES: GEO
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Project description:To characterize gene expression in spermatogenesis we sequenced eight mRNA samples from testes of juvenile mice aged between 6 and 38 days post partum. We then classified gene expression profiles and found over 1,000 meiotically-expressed protein-coding genes that have not been previously reported in relation to spermatogenesis. Furthermore, we developed an in silico de-convolution approach to estimate cell type-specific gene expression from temporal gene expression profiles and applied it to our dataset. To further characterize transcription during spermatogenesis, we determined RNA polymerase II (RNAPII) distribution along the genome using ChIP-Seq. mRNA sequencing at 8 time points during spermatogenesis was used to characterize differential gene expression; RNA Pol II ChIP-Seq at two time points was used to estimate RNA Pol II accumulation at promoters.
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Project description:The Senegalese sole (Solea senegalensis) is a marine flatfish of high economic value and a target species for aquaculture. Here, we used a transcriptomic approach to investigate changes in genes expressed in the Senegalese sole testis throughout spermatogenesis in wild-caught fish adapted to captivity. We identified approximately 400 genes that are differentially expressed during the progression of spermatogenesis and that participate in processes such as activation of the ubiquitin-proteasome system, sperm maturation and motility, cell adhesion or cytoskeletal remodeling. The results from this study contribute to our understanding of the molecular changes ocurring during spermatogenesis in the Senegalese sole. This study represents spermatogenesis in Solea senegalensis: early versus late spermatogenesis. Total RNA from testes at different stages in spermatogenesis (early, mid, late and functional maturation) from F0 wild Senegalese sole (3-4 animals at each stage) was extracted using the RNeasy extraction kit (Qiagen) and treated with DNAse following the manufacturer’s instructions. Quantitative and qualitative analysis of total RNA was performed using the Agilent 2100 bioanalyzer. RNA samples from each stage were pooled and amplified, labelled and hybridized to a custom-made oligonucleotide microarray containing 5,087 Senegalese sole Unigene sequences. In brief, pooled testicular RNAs from each stage were amplified and the resulting cRNAs labelled with Cy3 and Cy5, respectively, mixed in equal amounts and hybridized to the microarray for 17 h at 60 ºC. Each hybridization was performed at least in duplicate.
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