Transcriptome analysis of gene expression in young and aged oocyte samples (RNA-Seq)

The purpose of this study are to compare transcriptome profiling (RNA-seq) data in young and aged oocytes,and to evaluate the transcriptome alterataions after rapamycin treatment in aged oocytes. The sequencing libraries from young, aged and rapa-treated oocytes were generated according to protocols of Smart-seq2.Then cDNA libraries were checked on a Bioanalyzer instrument (Agilent) and the passed samples were finally sequenced by Illumina novaseq for 150-bp paired-end reads. RNA-seq data were aligned to the mouse genome mm10 using STAR 2-pass mode with the annotations of Ensembl version 90 and parameter '--outFilterMismatchNmax 6'. SAMtools was used to extract uniquely mapped reads with mapping quality more than or equal to 20. The number of reads mapped to each gene was counted using the HTSeq python package with the 'union' overlap resolution mode and –stranded = no. The expressions of genes were quantified as RPKM by edgeR . Genes with RPKM more than 1 in at least three control samples were considered as expressed. Only expressed genes were kept for the subsequent analysis. Differentially expressed genes were identified by DESeq2 using two criteria: fold change of expression should be higher than 2 and the adjusted p-value should be lower than 0.05. Enriched GO terms were obtained with P-value less than 0.05 from DAVID.The analysis of data revealed a total of 454 genes were significant differentially expressed during oocyte aging,of which, 242 genes were upregulated and 212 downregulated in aged oocytes.GO enrichment analysis showed that some of gene expressions were related to oxidation-reduction process, cell cycle, translation and mitochondrial functions. However, gene expression analysis revealed only 4 differently expressed genes between oocyte from rapa-treated and reproductively aged mice. Overall design: Total 9 samples from 4-week-old control, 48-week-old aged and 48-week-old (about 12 months) rapa-treated oocytes in 3 separate experiments, each sample contained about 10 oocytes.