Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome profiling of mutant embryos form the Mouse Genetics Project DMDD

ABSTRACT: The DMDD Programme (Deciphering the Mechanisms of Developmental Disorders) provides a free online database of morphological and molecular phenotypes from embryonic-lethal mouse gene knockouts (http://www.dmdd.org.uk/). Embryos are imaged using HREM, placentas are examined by histology and mutant embryo mRNA expression profiles are compared to wild type. Total RNA was extracted from somite number staged homozygous mutant, heterozygous and sibling wild-type E9.5 whole C57BL/6N embryos and DNase treated. The embryos were derived from Mouse Genetics Programme gene knockout lines defined as homozygous embryonic lethal (http://www.sanger.ac.uk/science/collaboration/mouse-resource-portal). Stranded RNA-seq libraries were constructed using the Illumina TruSeq Stranded RNA protocol with oligo dT pulldown.
Notes about samples and libraries:

(1) The knockout alleles in this project were: Cnot4: Cnot4^tm1b(EUCOMM)Wtsi ; Ssr2: Ssr2^tm1b(EUCOMM)Wtsi ; 4933434E20Rik: 4933434E20Rik^tm1a(EUCOMM)Wtsi ; 1700007K13Rik: 1700007K13Rik^tm2b(EUCOMM)Wtsi ; Camsap3: Camsap3^tm1a(EUCOMM)Wtsi ; Anks6: Anks6^tm1b(KOMP)Wtsi ; Pdzk1: Pdzk1^tm2b(EUCOMM)Wtsi ; Otud7b: Otud7b^tm1b(EUCOMM)Wtsi ; Adamts3: Adamts3^tm1b(KOMP)Wtsi; Mir96: Mir96^tm2.2WTSI ; Cyp11a1: Cyp11a1^tm1b(EUCOMM)Hmgu and Traf6: Traf6^tm2a(EUCOMM)Wtsi.

(2) A combination of litter identifier and embryo identifier within a litter will unambiguously identify a single embryo used in this study.

(3) There is a margin of error for the somite-stage information (+/- 1 somite) because some embryos could be in between somite stages.

(4) There are a minimum of three biological replicates per line.

(5) Sex of the embryos was determined post-RNA-seq by looking at the expression of Xist (strong expression in females only).

(6) Libraries were constructed in batches comprising multiple litters from one line from RNA-extraction to sequencing.

This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/ .

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Mus musculus

SUBMITTER: John Collins

PROVIDER: E-MTAB-5224 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The DMDD Programme (Deciphering the Mechanisms of Developmental Disorders, https://dmdd.org.uk/) provides a free online database of morphological and molecular phenotypes from embryonic-lethal mouse gene knockouts (http://www.dmdd.org.uk/). Embryos are imaged using HREM, placentas are examined by histology and mutant embryo mRNA expression profiles are compared to wild type. To underpin these investigations we have produced a comprehensive time series of gene expression through normal embryo development. Total RNA was extracted from somite number staged, second generation genotypically wild type, C57BL/6N embryos of mixed G0 linages from the Mouse Genetics Programme (http://www.sanger.ac.uk/science/collaboration/mouse-resource-portal) and DNase treated. Stranded RNA-seq libraries were constructed using the Illumina TruSeq Stranded RNA protocol with oligo dT pulldown. Notes about samples and libraries: (1) A combination of litter identifier and embryo identifier within a litter will unambiguously identify a single embryo used in this study. (2) There is a margin of error for the somite-stage information (+/- 1 somite) because some embryos could be in between somite stages. (3) All knockouts in the parents or grandparents are for embryonic lethal genes. (4) There are four biological replicates per somite-stage, with the exception of the 4-somite embryos (three biological replicates only). Altogether 111 embryos were sourced, of which 14 were sequenced twice to generate enough read depth/coverage. No new libraries were prepared for the repeated sequencing, despite the assays being assigned new ERX* accessions. (5) Sex of the embryos was determined post-RNA-seq by looking at the expression of Xist (strong expression in females only). (6) Library constructon batch refers to batch of sample handling post RNA-extraction (size selection, PCR amplification during library preparataion). (7) Superbatch gathers 17 representative embryos out of the 111 embryos post RNA-extraction, and put them through the library construction pipeline as one single batch. The generated data is intended to be used for normalisation using the remove unwanted variation (RUV) method. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/ .

Project description:Stem cell differentiation and lineage specification depend on coordinated programs of gene expression, but our knowledge of the chromatin modifying factors regulating these events remains incomplete. Ubiquitination of histone H2A (H2A-K119u) is a common chromatin modification associated with gene silencing, and controlled by the ubiquitin-ligase polycomb repressor complex 1 (PRC1) and H2A-deubiquitinating enzymes (H2A-DUBs). The roles of H2A-DUBs in mammalian development, stem cells, and haematopoiesis have not been addressed. Here we characterized an H2A-DUB targeted mouse line Mysm1-tm1a/tm1a and demonstrated defects in bone marrow haematopoiesis, resulting in lymphopenia, anemia, and thrombocytosis. Development of lymphocytes was impaired from the earliest stages of their differentiation; and there was also a depletion of erythroid cells and a defect in erythroid progenitor function. These phenotypes were due to a cell-intrinsic requirement for Mysm1 in the bone marrow. Importantly, Mysm1-tm1a/tm1a haematopoietic stem cells were functionally impaired, and this was associated with elevated levels of reactive oxygen species, γH2AX DNA damage marker, and p53 protein in the haematopoietic progenitors. Overall these data establish a role for Mysm1 in the maintenance of bone marrow stem cell function, in the control of oxidative stress and genetic stability in haematopoietic progenitors, and in the development of lymphoid and erythroid lineages. Total RNA from different mouse tissues (liver, bone marrow, brain) and cell types (embryonic fibroblasts - MEFs, and embryonic stem cells - ESCs) from wild type, Mysm1+/tm1a (heterozygous), and Mysm1tma1/tm1a (homozygous) mice was analyzed. Tissue and MEFs comparisons are between 3-4 animals per group; ESC comparisons are between three independently passaged samples of wild type, Mysm1+/tm1a, and Mysm1tma1/tm1aES-cells, all on C57BL/6 background. Full allele name: Mysm1tm1a(KOMP)WTSI . This Series includes the data from the tissues.

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Transcriptome profiling of mutant embryos form the Mouse Genetics Project DMDD

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