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Homo sapiens

ABSTRACT: Comprehensive evaluation of genome-wide 5-hydroxymethylcytosine profiling approaches in human DNA [BiSulfite-seq]

PROVIDER: PRJNA369396 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR5219980_1.fastq.gz	Fastqsanger.gz
	SRR5219980_2.fastq.gz	Fastqsanger.gz
	SRR5219981_1.fastq.gz	Fastqsanger.gz
	SRR5219981_2.fastq.gz	Fastqsanger.gz
	SRR5219982_1.fastq.gz	Fastqsanger.gz

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Comprehensive evaluation of genome-wide 5-hydroxymethylcytosine profiling approaches in human DNA

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Project description:High-resolution detection of genome-wide 5-hydroxymethylcytosine (5hmC) sites of small-scale samples represents a continuous challenge. Here, we present CATCH-seq, a bisulfite-free, base-resolution method for the genome-wide detection of 5hmC. CATCH-seq is based on selective 5hmC oxidation, labeling and subsequent C-to-T transition during PCR. Applications of CATCH-seq to nano-scale DNA samples reveal previously underappreciated non-CG 5hmCs in the hESC genome and base-resolution hydroxymethylome in human cell-free DNA.

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Precise genomic mapping of 5-hydroxymethylcytosine via covalent tether-directed sequencing

Project description:5-hydroxymethylcytosine (5hmC) is the most prevalent intermediate on the oxidative DNA demethylation pathway and is implicated in regulation of embryogenesis, neurological processes and cancerogenesis. Profiling of this relatively scarce genomic modification in clinical samples requires cost-effective high resolution techniques that avoid harsh chemical treatment. Here, we present a bisulfite-free approach for 5hmC profiling at single nucleotide resolution, named hmTOP-seq, which is based on direct sequence readout primed at covalently labeled 5hmC sites from an in situ tethered DNA oligonucleotide. Examination of distinct tethering chemistries suggested a putative model for the targeted non-homologous polymerase priming which permits theoretical evaluation of suitable tethers at the design stage. The robustness and resolution of hmTOP-seq was validated on a small model genome and mouse embryonic stem cells, which allowed construction of single nucleotide 5hmCG maps that reflect subtle differences in strand-specific CG hydroxymethylation. Collectively, hmTOP-seq expands the toolbox of valuable techniques for cost-effective identification of epigenetic changes and new disease biomarkers.

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Project description:We developed a novel approach, J-binding protein 1 sequencing (JBP1-seq), that combines the benefits of an improved recombinant JBP1 protein, Nextera-based library construction, and nextgeneration sequencing (NGS) for genome-wide profiling of 5-hydroxymethylcytosine (5hmC). Compared with the original JBP1, this new recombinant JBP1 was biotinylatedin vivo and conjugated to magnetic beads via biotin-streptavidin interactions. These modifications allowed a more efficient and consistent pull-down of β-glucosyl-5-hydroxymethylcytosine (β-glu-5hmC), and sequence-ready libraries can be generated within 4.5 hours from DNA inputs as low as 50 ng. 5hmC enrichment of human brain DNA using the new JBP1 resulted in over 25,000 peaks called, which is significantly higher than the 4,003 peaks enriched using the old JBP1. Comparison of the technical duplicates and validations with other platforms indicated the results are reproducible and reliable. Thus, JBP1-seq provides a fast, efficient, cost-effective method for accurate 5hmC genome-wide profiling. An improvement of JBP1-Seq

2014-11-26 | E-GEOD-61193 | biostudies-arrayexpress

Genome-wide Regulation of 5hmC, 5mC and Gene Expression by Tet1 Hydroxylase in Mouse Embryonic Stem Cells (bisulfite sequencing data)

Project description:DNA methylation of C5-cytosine (5mC) in the mammalian genome is a key epigenetic event that is critical for various cellular processes. However, how the genome-wide 5mC pattern is dynamically regulated remains a fundamental question in epigenetic biology. The TET family of 5mC hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), have provided a new potential mechanism for the dynamic regulation of DNA methylation. The extent to which individual Tet family members contribute to the genome-wide 5mC and 5hmC patterns and associated gene network remains largely unknown. Here we report genome-wide mapping of Tet1 and 5hmC in mESCs and reveal a mechanism of action by which Tet1 controls 5hmC and 5mC levels in mESCs. In combination with microarray and mRNA-seq expression profiling, we identify a comprehensive yet intricate gene network influenced by Tet1. We propose a model whereby Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting the existing 5mC to 5hmC through its enzymatic activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 target loci, thereby providing a new regulatory mechanism for establishing the epigenetic landscape of mESCs, which ultimately contributes to mESC differentiation and the onset of embryonic development. To determine the genome-wide DNA methylation changes caused by Tet1 depletion in mouse ES cells. Tet1 protein was depleted by specific siRNA treatment. The DNA methylation levels in control and Tet1 siRNA-transfected ES cells were determined by targeted bisulfite sequencing.

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