Pluto Bioinformatics

GSE129714: Transcriptome analysis of gene expression in control and Kiaa1429Zp3cKO samples (RNA-Seq)

Bulk RNA sequencing

The goals of this study are to compare transcriptome profiling (RNA-seq) data and quantitative reverse transcription polymerase chain reaction (qPCR) methods and to evaluate protocols for optimal high-throughput data analysis. The sequencing libraries from control and Kiaa1429Zp3cKO oocytes were generated according to protocols of Smart-seq2. For all samples the concentration of sequencing libraries was quantified using qPCR. All samples showed a concentration of higher than 3nM and were sequenced using HiSeq XTen (Illumina) with paired-end 150-bp sequencing.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 1081 genes were significant differentially expressed. 448 genes were upregulated and 633 downregulated in Kiaa1429Zp3cKO oocytes.We found that Kiaa1429 deletion altered expression pattern of many oocyte-derived factors (Ccnb1ip1, Lysmd2, Vav3, sohlh2, Id3, Anks4b, Scel, Cfap53, Olfm4, Fgf8, Susd3, Clip4, Fam124b, Gfra1, Nedd4l, Bmp15), which have been reported to be involved in signaling pathways of the oocyte-granulosa cell dialog and play important roles in oocyte survival and folliculogenesis. These oocyte-derived factors selected randomly were validated by qPCR in the GV oocytes from 20-day-old mice. SOURCE: Wenjie Shu (shuwj@bmi.ac.cn) - Beijing Institute of Radiation Medicine