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Total Stranded paired end RNA Seq of wildtype and knockout Kdm4a 2 cell embryos

ABSTRACT: Purpose: The goal of this study was to generate paired-end total RNA Seq transcriptomes of wildtype and Kdm4a 2 cell embryos to study the dynamics of differentially expressed repeats and transposons during early murine development Methods: 2 cell embryos cultured in KSOM EmbryoMax medium (Sigma) were subjected to the SMARTSeq Stranded Total RNASeq protocol according to the manufacturers instructions on the same day simultaneously for control and knockout embryos. Conclusions: Our study represents a detailed analysis of differentially expressed repeats and transposons in embryos lacking maternal or endogenous Kdm4a, with six biologic replicates, generated by SMARTSeq STranded paired end RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that there is a consistent downrgulation of specific LTR families afffected in most biological replicates, by the lack of Kdm4a giving us robust statistical significance. We conclude that Kdm4a is required for proper activation of these repeat elements in conjuction with a permissive chromatin landscape.

ORGANISM(S): Mus musculus

PROVIDER: GSE129733 | GEO | 2020/01/30

REPOSITORIES: GEO

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

Project description: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.

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Total Stranded paired end RNA Seq of wildtype and knockout Kdm4a 2 cell embryos

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