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Application of Microdroplet PCR for Large-Scale Targeted Bisulfite Sequencing

ABSTRACT: Cytosine methylation of DNA CpG dinucleotides in gene promoters is an epigenetic modification that regulates gene transcription. While many methods exist to interrogate methylation states, no current methods offer large-scale, targeted, single CpG resolution. We report an approach combining bisulfite treatment followed by RainDance microdroplet PCR with next-generation sequencing to assay the methylation state of 50 genes in the regions 1 kb upstream and downstream of their transcription start sites.

ORGANISM(S): Homo sapiens

PROVIDER: GSE30179 | GEO | 2011/06/25

SECONDARY ACCESSION(S): PRJNA143939

REPOSITORIES: GEO

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Application of Microdroplet PCR for Large-Scale Targeted Bisulfite Sequencing

Project description:Cytosine methylation of DNA CpG dinucleotides in gene promoters is an epigenetic modification that regulates gene transcription. While many methods exist to interrogate methylation states, no current methods offer large-scale, targeted, single CpG resolution. We report an approach combining bisulfite treatment followed by RainDance microdroplet PCR with next-generation sequencing to assay the methylation state of 50 genes in the regions 1 kb upstream and downstream of their transcription start sites. Wildtype and hypermethylated Jurkat DNA (New Englad Biolabs) was treated with bisulfite to convert all unmethylated cytosines to uracil. Following bisulfite treatment, targeted amplification was carried out using a custom primer library and microdroplet PCR. PCR product was sheared to 200 bp and ligated to sequencing adapters following standard protocols. Sequencing was conducted with single-end 100 bp reads on an Illumina GAIIx for wild type Jurkat DNA or Jurkat CpG DNA with a single sample per lane.

2011-06-25 | E-GEOD-30179 | biostudies-arrayexpress

Extended representation bisulfite sequencing of gene regulatory elements in multiplexed samples and single cells

Project description:DNA methylation of CpG dinucleotides impacts transcriptional activity and genome stability, and is altered in human disease. Whole genome and reduced-representation DNA methylation profiling methods have clarified biological roles of DNA methylation, but do not efficiently survey the vast numbers of noncoding regulatory elements in mammalian genomes.

2021-03-29 | GSE149954 | GEO

Application of microdroplet PCR for large-scale targeted bisulfite sequencing.

Project description:Cytosine methylation of DNA CpG dinucleotides in gene promoters is an epigenetic modification that regulates gene transcription. While many methods exist to interrogate methylation states, few current methods offer large-scale, targeted, single CpG resolution. We report an approach combining bisulfite treatment followed by microdroplet PCR with next-generation sequencing to assay the methylation state of 50 genes in the regions 1 kb upstream of and downstream from their transcription start sites. This method yielded 96% coverage of the targeted CpGs and demonstrated high correlation between CpG island (CGI) DNA methylation and transcriptional regulation. The method was scaled to interrogate the methylation status of 77,674 CpGs in the promoter regions of 2100 genes in primary CD4 T cells. The 2100 gene library yielded 97% coverage of all targeted CpGs and 99% of the target amplicons.

| S-EPMC3202290 | biostudies-literature

Reduced representation bisulfite sequencing (RRBS) of WT, Tet1-/-, Tet2-/-, Tet1-/-:Tet2-/- (DKO), and Tet1-/-:Tet2-/-:Tet3-/- (TKO) murine embryonic stem under normal culture conditions.

Project description:RRBS was used to investigate the methylation status of CpG dinucleotides in different Tet knockouts.

2019-06-26 | GSE122813 | GEO

Reduced representation bisulfite sequencing of equine tissue

Project description:The aim of the project was to identify the tissue-specific DNA methylation patterns of selected horse tissues, derived from two germ layers, endodermal (liver and lung) and mesenchymal (cardiac striated muscle) origin. The comparative analysis of DNA methylation patterns of the genome fraction rich in CpG dinucleotides was investigated using Reduced Representation Bisulfite Sequencing (RRBS) technique.

2022-12-31 | GSE191047 | GEO

Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain (Bisulfite-seq)

Project description:DNA methylation plays critical roles in the nervous system and has been traditionally considered to be restricted to CpG dinucleotides in metazoan genomes. Here we show that the single-base resolution neuronal DNA methylome from the adult mouse dentate gyrus consists of both CpG (~75%) and CpH (~25%) methylation (H = A/C/T). Neuronal CpH methylation is conserved in human brains, enriched in low CpG-density regions, depleted at protein-DNA interaction sites, and anti-correlated with gene expression. Functionally, both mCpGs and mCpHs can repress transcription in vitro and are recognized by MeCP2 in vivo. Unlike most CpG methylation, CpH methylation is established de novo during neuronal maturation and requires DNMT3A for active maintenance in post-mitotic neurons. These characteristics of CpH methylation suggest a significantly expanded proportion of the neuronal genome under cytosine methylation regulation and provide a new framework for understanding the roles of this key epigenetic modification in neuronal identity, maturation, plasticity and neurological disorders. Two biological replicates (dentate gyrus samples from C57Black6 mice) were analyzed in Bisulfite-seq.

2013-12-22 | E-GEOD-52330 | biostudies-arrayexpress

DNA Methylation by Reduced Representation Bisulfite Seq from ENCODE/HudsonAlpha

Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Florencia Pauli mailto:fpauli@hudsonalpha.org). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track is produced as part of the ENCODE project. The track reports the percentage of DNA molecules that exhibit cytosine methylation at specific CpG dinucleotides. In general, DNA methylation within a gene's promoter is associated with gene silencing, and DNA methylation within the exons and introns of a gene is associated with gene expression. Proper regulation of DNA methylation is essential during development and aberrant DNA methylation is a hallmark of cancer. DNA methylation status is assayed at more than 500,000 CpG dinucleotides in the genome using Reduced Representation Bisulfite Sequencing (RRBS). Genomic DNA is digested with the methyl-insensitive restriction enzyme MspI, small genomic DNA fragments are purified by gel electrophoresis, and then used to construct an Illumina sequencing library. The library fragments are treated with sodium bisulfite and amplified by PCR to convert every unmethylated cytosine to a thymidine while leaving methylated cytosines intact. The sequenced fragments are aligned to a customized reference genome sequence and for each assayed CpG we report the number of sequencing reads covering that CpG and the percentage of those reads that are methylated. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

2011-03-01 | GSE27584 | GEO

Whole genome bisulfite sequencing of Picocholorum soloecismus across several days of its growth cycle

Project description:Epigenetic modifications have not been well characterized in most microalgae species, particularly those used for biofuel production. We report the first analysis of DNA methylation of the species Picocholorum soloecismus across its growth cycle using whole-genome bisulfite sequencing. DNA methylation occurs in all three cytosine contexts (CpG, CHH, CHG) in P. soloecismus. While global DNA methylation is low overall (~1.15% of the 15 MB genome), methylation occurs appreciable quantities (12.1%) in CpG dinucleotides in a bimodal distribution. The P. soloecismus genome becomes hypomethylated during the growth cycle (across several days in culture). 5-aza-2’-deoxycytidine (5AZA) treatment was used to alter DNA methylation patterns, which resulted in altered algal physiology and site specific changes in CpG DNA methylation.

2020-08-01 | GSE155500 | GEO

Reduced representation bisulfite sequencing of equine sarcoid and tumor distant skin samples

Project description:The aim of the project was to identify sites undergoing changes in methylation level in cancer cells of equine sarcoid. The comparative analysis of DNA methylation patterns of the genome fraction rich in CpG dinucleotides was investigated using Reduced Representation Bisulfite Sequencing (RRBS) technique. In this study, for the first time we report the genome-wide DNA methylation profile of skin tumour in horses and describe differentially methylated genomic regions with respect to healthy skin.

2017-05-31 | GSE89859 | GEO

Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain (Bisulfite-seq)

2013-12-22 | GSE52330 | GEO

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