Project description:We describe the use of a novel DNA modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq) to map high-resolution hydroxymethylome of mouse E14 embryonic stem cells. The specificity of AbaSI enables sensitive detection of 5hmC at low occupancy regions. Bioinformatic analysis suggests 5hmCs in genic regions closely follows the 5mC distribution. 5hmC is generally depleted in CpG islands and only enriched in a small set of repetitive elements. A regularly spaced and oscillating 5hmC pattern was observed at the binding sites of CTCF. 5hmC is enriched at the poised enhancers with the mono-methylated histone H3 lysine 4 (H3K4me1) marks, but not at the active enhancers with the acetylated histone H3 lysine 27 (H3K27Ac) marks. Non-CG hydroxymethylation appears to be prevalent in the mitochondrial genome. We propose that some amounts of transiently stable 5hmCs may indicate a poised epigenetic state or de-methylation intermediate, while others may suggest a locally accessible chromosomal environment to the TET enzymatic apparatus. Mapping of genomic 5-hydroxymethylcytosine in mouse embryonic stem cell by enzymatic digistion of AbaSI coupled with high-throughput sequencing, in replicates.
Project description:We analyzed levels of 5-methyl cytosine nnnn CCCGGG target sites by sequential restriction digest by SmaI and XmaI enzymes, ligating Illumina adaptors to the restriction fragments and reading methylation-specific signatures at the ends of restriction fragments by paired ends Illumina high throughput sequencing. Digital restriction enzyme analysis of methylation (DREAM) was performed to determine the methylation profile of SW48 colon cancer cell line genomic DNA. Genomic DNA spiked in with unmethylated, partially methylated and fully methylated standards was sequentially cut at CCCGGG sites with the methylation-sensitive enzyme SmaI (blunt ends) and its methylation-tolerant neoschizomer XmaI (5'CCGG overhangs), creating different end sequences that represented methylation status of the CCCGGG sites. These end sequences were analyzed by Illumina high throughput sequencing. Methylation status at individual CCCGGG sites across the genome was determined by counting the methylated reads with the CCGGG signature and unmethylated reads with the GGG signature at the beginnings of the sequencing reads after alignment to the human genome.
Project description:We describe the use of a novel DNA modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq) to map high-resolution hydroxymethylome of mouse E14 embryonic stem cells. The specificity of AbaSI enables sensitive detection of 5hmC at low occupancy regions. Bioinformatic analysis suggests 5hmCs in genic regions closely follows the 5mC distribution. 5hmC is generally depleted in CpG islands and only enriched in a small set of repetitive elements. A regularly spaced and oscillating 5hmC pattern was observed at the binding sites of CTCF. 5hmC is enriched at the poised enhancers with the mono-methylated histone H3 lysine 4 (H3K4me1) marks, but not at the active enhancers with the acetylated histone H3 lysine 27 (H3K27Ac) marks. Non-CG hydroxymethylation appears to be prevalent in the mitochondrial genome. We propose that some amounts of transiently stable 5hmCs may indicate a poised epigenetic state or de-methylation intermediate, while others may suggest a locally accessible chromosomal environment to the TET enzymatic apparatus.
Project description:We analyzed levels of 5-methyl cytosine nnnn CCCGGG target sites by sequential restriction digest by SmaI and XmaI enzymes, ligating Illumina adaptors to the restriction fragments and reading methylation-specific signatures at the ends of restriction fragments by paired ends Illumina high throughput sequencing.
Project description:We analyzed levels of 5-methyl cytosine CCCGGG target sites by sequential restriction digest by SmaI and XmaI enzymes, ligating Illumina adaptors to the restriction fragments and reading methylation-specific signatures at the ends of restriction fragments by paired ends Illumina high throughput sequencing.
Project description:We analyzed levels of 5-methyl cytosine at CCCGGG target sites by sequential restriction digest by SmaI and XmaI enzymes, ligating Illumina adaptors to the restriction fragments and reading methylation-specific signatures at the ends of restriction fragments by paired ends Illumina high throughput sequencing.
Project description:We analyzed levels of 5-methyl cytosine at CCCGGG target sites by sequential restriction digest by SmaI and XmaI enzymes, ligating Illumina adaptors to the restriction fragments and reading methylation-specific signatures at the ends of restriction fragments by paired ends Illumina high throughput sequencing.
Project description:We analyzed levels of 5-methyl cytosine CCCGGG target sites by sequential restriction digest by SmaI and XmaI enzymes, ligating Illumina adaptors to the restriction fragments and reading methylation-specific signatures at the ends of restriction fragments by paired ends Illumina high throughput sequencing.
Project description:We analyzed levels of 5-methyl cytosine CCCGGG target sites by sequential restriction digest by SmaI and XmaI enzymes, ligating Illumina adaptors to the restriction fragments and reading methylation-specific signatures at the ends of restriction fragments by paired ends Illumina high throughput sequencing.
Project description:We analyzed levels of 5-methyl cytosine nnnn CCCGGG target sites by sequential restriction digest by SmaI and XmaI enzymes, ligating Illumina adaptors to the restriction fragments and reading methylation-specific signatures at the ends of restriction fragments by paired ends Illumina high throughput sequencing.