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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.

2020-01-30 | GSE129731 | GEO

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

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

| PRJNA532606 | ENA

Genome-wide maps of H3K36me3 in MII oocytes and H3K9me3 in 2-cell embryos

Project description:We report the application of low input high-throughput profiling of histone modifications in mouse oocytes and 2-cell embryos. We used antibodies against H3K36me3 (190 wildtype and 93 knockout MIIcoocytes pooled as one replicate) and H3K9me3 ( around 118 control and MZ embryos / replicate; 1 biological replicates per genotype). In the case of H3K36me3, we find that its establishment and maintence is not affected in oocytes lacking Kdm4a. In the case of H3K9me3 in 2-cell embryos, we find that the histone mark aberrantly gained in Kdm4a deficient oocytes is also maintained in the MZ mutant 2-cell embryo. This shows that Kdm4a is important to keep open chromatin clear of H3K9me3 spreading in oocytes in order to transfer a healthy epigenome amenable for proper genome activation post fertilization.

2020-01-30 | GSE138951 | GEO

2a0acda9-ec4c-41f1-9421-a257af2bee89 - samples

Project description:Tcells were isolated fom Tissues and FACS sorted. In total 43 samples were processed in 5 replicates with the Takara SmartSeq Stranded kit. Single end fastq-files are supplied.

| EGAD00001007664 | EGA

2b8073eb-290d-4809-ac68-e0d01100dc61 - samples

Project description:Tcells were isolated fom Tissues and FACS sorted. In total 50 samples were processed in 5 replicates with the Takara SmartSeq Stranded kit. Single end fastq-files are supplied.

| EGAD00001007663 | EGA

Genome-wide expression analysis in kyp and cmt3 mutants of Arabidopsis thaliana

Project description:Investigation of genome-wide expression in the mutant of histone H3K9 methyltransferase KRYPTONITE (KYP) or DNA methyltransferase CHROMOMETHYLASE3 (CMT3) in Arabidopsis. These mutants showed decrease in H3K9 methylation and DNA methylation levels, and transcriptional activation at transposons and repeats. Using NimbleGen DNA microarray, global pattern of expression of genes and transposons were examined in these mutants. Total RNA from leaves was isolated using the RNeasy Plant Mini kit (Qiagen) and was treated with DNase I (TAKARA). Double-stranded cDNA was synthesized using the SuperScript Double Stranded cDNA Synthesis kit (Invitrogen) and oligo (dT)20 primer. cDNA was labeled by Cy3, hybridized to 4 x 72k array, and scanned according to manufacture's instructions (www.nimblegen.com).

2010-09-21 | E-GEOD-22957 | biostudies-arrayexpress

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

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

| PRJNA532600 | ENA

HEK293 cells 100 cell RNAseq profiling on Ion Proton

Project description:Five libraries from 100 HEK293 cells each were prepared using a Smartseq based custom library preparation approach with unique molecular identifiers. One batch of 2 replicates (A) and one batch of 3 replicates (B) were prepared from different cell cultures. Libraries were sequenced on an Ion Proton

2016-12-19 | GSE79123 | GEO

Histone Demethylase-Assisted Somatic Cell Nuclear Transfer Facilitates Derivation of Human Pluripotent Stem Cells

Project description:The extremely low efficiency of human embryonic stem cell (hESC) derivation using somatic cell nuclear transfer (SCNT) limits potential application. Blastocyst formation from human SCNT embryos occurs at a low rate and with only some oocyte donors. We previously showed in mice that reduction of histone H3 lysine 9 trimethylation (H3K9me3) through ectopic expression of the H3K9me3 demethylase Kdm4d greatly improves SCNT embryo development. Here we show that overexpression of a related H3K9me3 demethylase KDM4A improves human SCNT, and that, as in mice, H3K9me3 in the human somatic cell genome is an SCNT reprogramming barrier. Overexpression of KDM4A significantly improves the blastocyst formation rate in human SCNT embryos by facilitating transcriptional reprogramming, allowing derivation of NTESCs from all oocyte donors tested using adult AMD patient somatic nuclei donors. This conserved mechanistic insight has potential applications for improving SCNT in a variety of contexts, including regenerative medicine. Here we perform RNA-seq based transcriptome profiling in human Donor (fibroblast cells), in vitro fertilized embryos at 8-cell stages (IVF_8Cell), somatic cell nuclear transfer embryos at 8-cell stages (SCNT_8Cell), SCNT assisted by KDM4A 8-cell embryos (SCNT_KDM4A_8Cell). Besides, we also perform RNA-seq in Control human ES cells (CTR_hES) and SCNT assisted by KDM4A derived human ES cells (NTK) with duplicates.Â

2016-03-01 | E-GEOD-73362 | biostudies-arrayexpress

Genome-wide maps of H3K4me3, H3K9me3 chromatin states in control and Kdm4a knockout oocytes

Project description:We report the application of low input high-throughput profiling of histone modifications in mouse oocytes. Using antibodies against H3K4me3 (100 oocytes/replicate; 2 biological replicates per genotype) and H3K9me3 ( around 300 oocytes/replicate; 2 biological replicates per genotype), we find that they are mutually exclusive in oocytes and this property is lost in oocytes lacking Kdm4a. H3K9me3 is spread into regions of H3K4me3 postive open chromatin which is surprisingly, well preserved leading to a potential bivalency of scale. Altogether, KDM4A is important to keep H3K4me3 marked open chromatin clear of H3K9me3 spreading in oocytes.

2020-01-30 | GSE129729 | GEO

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