Pluto Bioinformatics

GSE129731: SMARTSeq_v4 paired end mRNA Seq of Kdm4a control mutant oocytes and preimplantation embryos

Bulk RNA sequencing

Purpose: The goal of this study was to generate paired-end mRNA Seq transcriptomes of control and Kdm4a mutant oocytes, 2-cell, 4-cell and 8-cell preimplantation embryos to study the dynamics of differentially expressed genes during early development; Methods: MII oocytes were isolated from wildtype and Kdm4a knockout mice. 16 MII oocytes from each genotype were washed in M2 medim after zona pellucida removal, washed once in nuclease free water and directly lysed and cDNA prepared according to the manufacturers instructions. Meanwhile, control and knockout MII oocytes were also in vitro fertilised with Kdm4a knockout sperm to produce control (+/-) and maternal mutant (-/-) embryos, and cultured in KSOM EmbryoMax medium (Sigma). 16 embryos were staged each day and harvested according to control embryos. Healthiest mutants were chosen for comparison with minimal secondary effects arising from dead/dying embryos. Similar to oocytes, individual cDNA were prepared and amplified according to manufacturers instructions in nuclease free conditions.; Conclusions: Our study represents a detailed analysis of differentially expressed genes in oocytes and embryos lacking maternal and endogenous Kdm4a, with sixteen biological replicates, generated by SMARTSeq v4 paired end RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles between the samples at each developmental stage. Our results show that there is a consistent downrgulation of zygotic genome activation (ZGA) genes in the mutant 2-cell embryos. Mutant 4 and 8-cell embryos have significant upregulation of minor ZGA genes giving us robust statistical significance. We conclude that Kdm4a is required for proper gene activation during maternal-to zygotic transition in conjuction with a permissive chromatin landscape. SOURCE: Aditya Sankar ( - Hoffmann University of Copenhagen

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