Single-CpG resolution mapping of 5-hydroxymethylcytosine by chemical labeling and exonuclease digestion (SCL-exo)
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE70635 | GEO | 2016/03/04
SECONDARY ACCESSION(S): PRJNA289245
REPOSITORIES: GEO
Similar Datasets
Project description:We present here a novel approach called Reduced Representation 5-Hydroxymethylcytosine Profiling (RRHP), which exploits ?-glucosyltransferase (?-GT) to inhibit restriction digestion at adapters ligated to a genomic library, such that only fragments presenting glucosylated 5hmC residues at adapter junctions will be amplified and sequenced. This assay profiles 5hmC sites with single-base resolution in a strand-specific manner. The absence of harsh chemical conversion steps allows for sequencing of native DNA with less inputs, enhancing both sequencing quality and mapping efficiency. Most importantly, the method proves highly reproducible and is a positive display method, sensitive enough to interrogate 5hmC sites with low abundance. When combined with existing RRBS data, it allows simultaneous comparison of 5mC and 5hmC at specific site. developing a new assay for genomic profiling of 5hmC
2014-08-26 | E-GEOD-49546 | biostudies-arrayexpress
Project description:We present here a novel approach called Reduced Representation 5-Hydroxymethylcytosine Profiling (RRHP), which exploits β-glucosyltransferase (β-GT) to inhibit restriction digestion at adapters ligated to a genomic library, such that only fragments presenting glucosylated 5hmC residues at adapter junctions will be amplified and sequenced. This assay profiles 5hmC sites with single-base resolution in a strand-specific manner. The absence of harsh chemical conversion steps allows for sequencing of native DNA with less inputs, enhancing both sequencing quality and mapping efficiency. Most importantly, the method proves highly reproducible and is a positive display method, sensitive enough to interrogate 5hmC sites with low abundance. When combined with existing RRBS data, it allows simultaneous comparison of 5mC and 5hmC at specific site.
2014-08-26 | GSE49546 | GEO
Project description:5-Methylcytosine (5-mC) is an important DNA modification found in eukaryotes that impacts gene regulation and disease pathogenesis. Recently, 5-hydroxymethylcytosine (5-hmC), another form of DNA modification, has been identified in substantial amounts in certain mammalian cell types; however, its roles as well as its distribution in mammalian genomes are unknown. Here we present a selective chemical labeling method for 5-hmC by utilizing T4 bacteriophage BGT-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC, which in turn can chemically incorporate a biotin group for detection, affinity enrichment, and sequencing of 5-hmC in mammalian genomes. Using this highly effective method, we demonstrate that 5-hmC is present in human cell lines beyond those previously recognized. We also find a gene expression level-dependent enrichment of intragenic 5-hmC in mouse cerebellum and an age-dependent acquisition of this modification in specific gene bodies linked to neurodegenerative disorders Identification of 5hmC enriched genmoic regions in mouse cerebellum
2010-12-12 | E-GEOD-25398 | biostudies-arrayexpress
Project description:DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays critical roles in a variety of biological and pathological processes in mammals. In active DNA demethylation, the ten-eleven translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution are still highly desirable. Herein, we propose a Chemical Labeling Enrichment and Deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by A3A and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mouse embryonic stem cells. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution.
2023-08-31 | GSE239943 | GEO
Project description:Mapping genome-wide 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) at single-base resolution is important to understand their biological functions. We present a cost-efficient mapping method that combines 5hmC-specific restriction enzyme PvuRts1I with a 5hmC enrichment method. The sensitive method enables detection of low abundant 5hmC sites, providing a more complete 5hmC landscape than available bisulfite-based methods. This method generated the first genome-wide 5fC map at single-base resolution. Parallel analyses revealed that 5hmC and 5fC existed with lower abundance and more dynamically in non-CpG context than in CpG context. In the genic region, distribution of 5hmCpG and 5fCpG differed from 5hmCH and 5fCH (H=A, T, C). 5hmC and 5fC were distributed distinctly at regulatory protein-DNA binding sites, depleted in permissive transcription factor binding sites, and enriched at active and poised enhancers. This sensitive bisulfite-conversion free method can be applied to biological samples with limited starting material or low abundance of cytosine modifications. Sensitive mapping of genome-wide 5-hydroxymethylcytosine and 5-formylcytosine in mouse embryonic stem cell at single-base resolution by combining 5-hydroxymethylcytosine specific restriction enzyme PvuRts1I and 5-hydroxymethylcytosine enrichment method (selective chemical labeling or SEAL)
2014-12-22 | E-GEOD-64186 | biostudies-arrayexpress
Project description:We generated a genome-wide map of 5hmC in rice panicle using a sensitive chemical labeling method to capture DNA fragments with 5hmC modification, followed by NGS sequencing. We showed that the 5hmC peaks distribution is non-random, about 52% 5hmC was located in gene bodies, 25% in intergenic regions, 11% in promoters, and 12% in immediate downstream. It was significantly enriched in heterochromatic regions of chromosomes 4 and 10, and especially located around TE genes. Combined with RNA-seq transcriptome data, we found that the gene-specific alterations of 5hmC can result in gene activation or repression, suggesting a potential role for 5hmC in gene regulation. From the compared distribution analysis among super-hybrid rice cultivars LYP9, and its parental lines 93-11 and PA64s, we investigated the cultivar-specificity of both the global levels and locus-specific distribution of 5hmC. Our data provided an overview of the genomic distribution of 5hmC in rice panicle. This will set the stage for further functional characterization of this novel DNA modification and its biosynthesis in rice. Examination of 5hmC modifications in rice panicle This submission represents ChIP-Seq component of study.
2014-04-15 | E-GEOD-37242 | biostudies-arrayexpress
Project description:We generated a genome-wide map of 5hmC in rice panicle using a sensitive chemical labeling method to capture DNA fragments with 5hmC modification, followed by NGS sequencing. We showed that the 5hmC peaks distribution is non-random, about 52% 5hmC was located in gene bodies, 25% in intergenic regions, 11% in promoters, and 12% in immediate downstream. It was significantly enriched in heterochromatic regions of chromosomes 4 and 10, and especially located around TE genes. Combined with RNA-seq transcriptome data, we found that the gene-specific alterations of 5hmC can result in gene activation or repression, suggesting a potential role for 5hmC in gene regulation. From the compared distribution analysis among super-hybrid rice cultivars LYP9, and its parental lines 93-11 and PA64s, we investigated the cultivar-specificity of both the global levels and locus-specific distribution of 5hmC. Our data provided an overview of the genomic distribution of 5hmC in rice panicle. This will set the stage for further functional characterization of this novel DNA modification and its biosynthesis in rice.
2014-04-15 | GSE37242 | GEO
Project description:While the role of 5-methylcytosine has been well studied, the biological role of 5-hydroxymethylcytosine still remains unclear due to the limited methods available for single-base detection of 5-hydroxymethylcytosine (5hmC). Here we present Mirror bisulfite sequencing for the detection of 5hmC at a single CpG site by synthesizing a DNA strand to mirror the parental strand. This semi-conservative duplex is sequentially treated with β-glucosyltransferase and M.SssI methylase. A glucosyl-5hmCpG in the parental strand inhibits methylation of the mirroring CpG site, and after bisulfite conversion, a thymine in the mirroring strand indicates a 5hmCpG site in the parental strand whereas a cytosine indicates a non-5hmC site. Using this method, the 5hmC levels of various human tissues and paired tumor liver tissues were mapped genome-wide.
2018-05-22 | GSE69272 | GEO
Project description:The study of 5-hydroxylmethylcytosines (5hmC), the sixth base of the mammalian genome, as an epigenetic mark has been hampered by a lack of method to map it at single-base resolution. Previous affinity purification-based methods could not precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach for mapping 5hmC at base resolution. Application of this new method to the embryonic stem cells not only confirms widespread distribution of 5hmC in mammalian genome, but also reveals a strong sequence bias and strand asymmetry at sites of 5hmC. Additionally, the relative abundance of 5hmC varies significantly depending on the types of functional sequences, suggesting different mechanisms for 5hmC deposition and maintenance. Furthermore, we observe high levels of 5hmC and reciprocally low levels of 5mC at transcription factor binding sites, revealing a dynamic DNA methylation process at cis-regulatory elements. Base resolution sequencing of 5 hydroxymethylcytosine in human and mouse embryonic stem cells
2012-05-16 | E-GEOD-36173 | biostudies-arrayexpress
Project description:Mapping genome-wide 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) at single-base resolution is important to understand their biological functions. We present a cost-efficient mapping method that combines 5hmC-specific restriction enzyme PvuRts1I with a 5hmC enrichment method. The sensitive method enables detection of low abundant 5hmC sites, providing a more complete 5hmC landscape than available bisulfite-based methods. This method generated the first genome-wide 5fC map at single-base resolution. Parallel analyses revealed that 5hmC and 5fC existed with lower abundance and more dynamically in non-CpG context than in CpG context. In the genic region, distribution of 5hmCpG and 5fCpG differed from 5hmCH and 5fCH (H=A, T, C). 5hmC and 5fC were distributed distinctly at regulatory protein-DNA binding sites, depleted in permissive transcription factor binding sites, and enriched at active and poised enhancers. This sensitive bisulfite-conversion free method can be applied to biological samples with limited starting material or low abundance of cytosine modifications.
2014-12-22 | GSE64186 | GEO