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Large conserved domains of low DNA methylation maintained by 5-hydroxymethycytosine and Dnmt3a [RNA-Seq]

ABSTRACT: To investigate the global transcriptome expression in Wild Type mouse hematopoietic stem cells, we performed high-throughput sequencing of Poly A+ RNA (RNA-Seq) from purified HSCs (SP-KSL-CD150+). With biological duplicates, more than 200 million reads in total were obtained.

ORGANISM(S): Mus musculus

PROVIDER: GSE49715 | GEO | 2013/11/16

SECONDARY ACCESSION(S): PRJNA214818

REPOSITORIES: GEO

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Large conserved domains of low DNA methylation maintained by 5-hydroxymethycytosine and Dnmt3a [RNA-Seq]

Project description:To investigate the global transcriptome expression in Wild Type mouse hematopoietic stem cells, we performed high-throughput sequencing of Poly A+ RNA (RNA-Seq) from purified HSCs (SP-KSL-CD150+). With biological duplicates, more than 200 million reads in total were obtained. Mouse hematopoietic stem cell mRNA profiles of wild type mice were generated by deep sequencing, in duplicate, using Illumina Hiseq 2000.

2013-11-16 | E-GEOD-49715 | biostudies-arrayexpress

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Large conserved domains of low DNA methylation maintained by 5-hydroxymethycytosine and Dnmt3a [Bisulfite-Seq]

Project description:Gains and losses in DNA methylation are prominent genomic features of all mammalian cell types. To gain insight into mechanisms that could promote shifts in DNA methylation patterns and thus contribute to cell fate, including malignant transformation, we performed genome-wide mapping of 5-methylcytosine in purified wild type and Dnmt3a conditional knockout hematopoietic stem cells (HSCs). We generated 1,121 million (control HSCs), and 1,213 million (Dnmt3a knockout HSCs) raw reads; about 81.4% and 88.7%, respectively, were successfully aligned to either strand of the reference genome (mm9), obtainig an average CG coverage of 39.5-fold (wild type) and 47.0-fold (Dnmt3a kockout). Whole genome bisulfite sequencing of secondarily-transplanted wild-type and Dnmt3a conditional knockout hematopoietic stem cells using Illumina HiSeq 2000

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Large conserved domains of low DNA methylation maintained by Dnmt3a.

Project description:Gains and losses in DNA methylation are prominent features of mammalian cell types. To gain insight into the mechanisms that promote shifts in DNA methylation and contribute to changes in cell fate, including malignant transformation, we performed genome-wide mapping of 5-methylcytosine and 5-hydroxymethylcytosine in purified mouse hematopoietic stem cells. We discovered extended regions of low methylation (canyons) that span conserved domains frequently containing transcription factors and are distinct from CpG islands and shores. About half of the genes in these methylation canyons are coated with repressive histone marks, whereas the remainder are covered by activating histone marks and are highly expressed in hematopoietic stem cells (HSCs). Canyon borders are demarked by 5-hydroxymethylcytosine and become eroded in the absence of DNA methyltransferase 3a (Dnmt3a). Genes dysregulated in human leukemias are enriched for canyon-associated genes. The new epigenetic landscape we describe may provide a mechanism for the regulation of hematopoiesis and may contribute to leukemia development.

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Large conserved domains of low DNA methylation maintained by 5-hydroxymethycytosine and Dnmt3a [Bisulfite-Seq]

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Large conserved domains of low DNA methylation maintained by 5-hydroxymethycytosine and Dnmt3a (CMS bisulfite-seq)

Project description:Gains and losses in DNA methylation are prominent genomic features of all mammalian cell types. To gain insight into mechanisms that could promote shifts in DNA 5-hydroxymethylation (5hmc) patterns and thus contribute to cell fate, including malignant transformation, we performed genome-wide mapping of 5hmc in purified wild type and Dnmt3a conditional knockout hematopoietic stem cells (HSCs) using cytosine-5-methylenesulphonate sequencing (CMS-seq). Comparing anti-CMS pulldown to input control, we identified 107,549 and 175,682 peaks of 5hmc enrichment in control and Dnmt3a knockout HSCs, respectively. Whole genome CMS-enriched bisulfite sequencing of secondarily-transplanted wild-type and Dnmt3a conditional knockout hematopoietic stem cells using Illumina HiSeq 2000

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Large conserved domains of low DNA methylation maintained by 5-hydroxymethycytosine and Dnmt3a (CMS bisulfite-seq)

Project description:Gains and losses in DNA methylation are prominent genomic features of all mammalian cell types. To gain insight into mechanisms that could promote shifts in DNA 5-hydroxymethylation (5hmc) patterns and thus contribute to cell fate, including malignant transformation, we performed genome-wide mapping of 5hmc in purified wild type and Dnmt3a conditional knockout hematopoietic stem cells (HSCs) using cytosine-5-methylenesulphonate sequencing (CMS-seq). Comparing anti-CMS pulldown to input control, we identified 107,549 and 175,682 peaks of 5hmc enrichment in control and Dnmt3a knockout HSCs, respectively.

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Controlling Genetic Heterogeneity in Gene-edited Hematopoietic Stem Cells by Single Cell Expansion

Project description:Gene editing using engineered nucleases frequently produces unintended genetic lesions in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain heterogenous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201+CD150+CD48-c-Kit+Sca-1+Lin-(KSL) population of pre-cultured HSCs. This dataset compares the gene expression in three different populations: (1) CD201+CD150+CD48-KSL (2) CD201+CD150+CD48+KSL and (3) CD201-KSL cells.

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