Extended representation bisulfite sequencing of gene regulatory elements in multiplexed samples and single cells
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ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE149954 | GEO | 2021/03/29
REPOSITORIES: GEO
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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.
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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.
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Project description:Sequencing based approaches have led to new insights about DNA methylation. While many different techniques for genome-scale mapping of DNA methylation have been employed, throughput has been a key limitation for most. To further facilitate the mapping of DNA methylation, we describe a protocol for gel-free multiplexed reduced representation bisulfite sequencing (mRRBS) that reduces the workload dramatically and enables processing of 96 or more samples per week. mRRBS achieves similar CpG coverage as the original RRBS protocol, while the higher throughput and lower cost make it better suited for large-scale DNA methylation mapping studies including cohorts of cancer samples. Libraries of 96 human samples
2012-08-29 | E-GEOD-40429 | biostudies-arrayexpress
Project description:Sequencing based approaches have led to new insights about DNA methylation. While many different techniques for genome-scale mapping of DNA methylation have been employed, throughput has been a key limitation for most. To further facilitate the mapping of DNA methylation, we describe a protocol for gel-free multiplexed reduced representation bisulfite sequencing (mRRBS) that reduces the workload dramatically and enables processing of 96 or more samples per week. mRRBS achieves similar CpG coverage as the original RRBS protocol, while the higher throughput and lower cost make it better suited for large-scale DNA methylation mapping studies including cohorts of cancer samples.
2012-08-29 | GSE40429 | GEO
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
Project description:Reduced Representation Bisulfite Sequencing was used to assess that changes in intestinal DNA methylation associated with NEC outbreak.
2020-12-01 | GSE149403 | GEO
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 DNA methylation at CpG sites was assayed with a modified version of Reduced Representation Bisulfite Sequencing (RRBS; Meissner et al., 2008). RRBS was performed on cell lines grown by many ENCODE production groups. The production group that grew the cells and isolated genomic DNA is indicated in the "obtainedBy" field of the metadata. When a cell type was provided by more than one lab, the data for the cells from only one lab are displayed in the table above. However, the data for every cell type from every lab is available from the Downloads page. RRBS was carried out by the Myers production group at the HudsonAlpha Institute for Biotechnology. Isolation of genomic DNA Genomic DNA is isolated from biological replicates of each cell line using the QIAGEN DNeasy Blood & Tissue Kit according to the instructions provided by the manufacturer. DNA concentrations for each genomic DNA preparation are determined using fluorescent DNA binding dye and a fluorometer (Invitrogen Quant-iT dsDNA High Sensitivity Kit and Qubit Fluorometer). Typically, 1 µg of DNA is used to make an RRBS library; however, we have also had success in making libraries with 200 ng genomic DNA from rare or precious samples. RRBS library construction and sequencing RRBS library construction starts with MspI digestion of genomic DNA , which cuts at every CCGG regardless of methylation status. Klenow exo- DNA Polymerase is then used to fill in the recessed end of the genomic DNA and add an adenosine as a 3prime overhang. Next, a methylated version of the Illumina paired-end adapters is ligated onto the DNA. Adapter ligated genomic DNA fragments between 105 and 185 basepairs are selected using agarose gel electrophoresis and Qiagen Qiaquick Gel Extraction Kit. The selected adapter-ligated fragments are treated with sodium bisulfite using the Zymo Research EZ DNA Methylation Gold Kit, which converts unmethylated cytosines to uracils and leaves methylated cytosines unchanged. Bisulfite treated DNA is amplified in a final PCR reaction which has been optimized to uniformly amplify diverse fragment sizes and sequence contexts in the same reaction. During this final PCR reaction uracils are copied as thymines resulting in a thymine in the PCR products wherever an unmethylated cytosine existed in the genomic DNA. The sample is now ready for sequencing on the Illumina sequencing platform. These libraries were sequenced with an Illumina Genome Analyzer IIx according to the manufacturer's recommendations. Data analysis To analyze the sequence data, a reference genome is created that contains only the 36 base pairs adjacent to every MspI site and every C in those sequences is changed to T. A converted sequence read file is then created by changing each C in the original sequence reads to a T. The converted sequence reads are aligned to the converted reference genome, and only reads that map uniquely to the reference genome are kept. Once reads are aligned the percent methylation is calculated for each CpG using the original sequence reads. The percent methylation and number of reads is reported for each CpG.
2011-03-01 | E-GEOD-27584 | biostudies-arrayexpress
Project description:DNA methylation is an important regulatory mechanism that contributes to transcriptional repression. To examine whether DNA methylation affects the transcriptional changes of DNA repair genes under H2O2-induced oxidative stress, we performed reduced representation bisulfite sequencing (RRBS) in H2O2-treated and untreated HCT116 cells.
2016-05-01 | GSE75963 | GEO
Project description:DNA methylation and the Polycomb Repression System are epigenetic mechanisms that play important roles in maintaining transcriptional repression. Recent evidence suggests that DNA methylation can attenuate the binding of Polycomb protein components to chromatin and thus plays a role in determining their genomic targeting. However, whether this role of DNA methylation is important in the context of transcriptional regulation is unclear. By genome-wide mapping of the Polycomb Repressive Complex 2 (PRC2)-signature histone mark, H3K27me3, in severely DNA hypomethylated mouse somatic cells, we show that hypomethylation leads to widespread H3K27me3 redistribution, in a manner that reflects the local DNA methylation status in wild-type cells. Unexpectedly, we observe striking loss of H3K27me3 and PRC2 from Polycomb-target gene promoters in DNA hypomethylated cells, including Hox gene clusters. Importantly, we show that many of these genes become ectopically expressed in DNA hypomethylated cells, consistent with loss of Polycomb-mediated repression. An intact DNA methylome is required for appropriate Polycomb-mediated gene repression by constraining PRC2 targeting. These observations identify a previously unappreciated role for DNA methylation in gene regulation and therefore influence our understanding of how this epigenetic mechanism contributes to normal development and disease. comparison of Dnmt1+/+ vs Dnmt1-/- mouse embryonic fibroblasts by Reduced Representation Bisulfite Sequencing
2013-03-01 | E-GEOD-44760 | biostudies-arrayexpress
Project description:DNA methylation is a mechanism for long-term transcriptional regulation and is required for normal cellular differentiation. Failure to properly establish or maintain DNA methylation patterns leads to cell dysfunction and diseases such as cancer. Identifying DNA methylation signatures in complex tissues can be challenging due to inaccurate cell enrichment methods and low DNA yields. We have developed a technique called Laser Capture Microdissection-Reduced Representation Bisulfite Sequencing (LCM-RRBS) for the multiplexed interrogation of the DNA methylation status of CpG Islands and promoters. LCM-RRBS accurately and reproducibly profiles genome-wide methylation of DNA extracted from microdissected fresh frozen or formalin-fixed paraffin-embedded tissue samples. To demonstrate the utility of LCM-RRBS, we characterized changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse. Compared to adjacent normal tissue, the adrenocortical tumors showed reproducible gains and losses of DNA methylation at genes involved in cell differentiation and organ development. LCM-RRBS is a rapid, cost-effective, and sensitive technique for analyzing DNA methylation in heterogeneous tissues and will facilitate the investigation of DNA methylation in cancer and organ development. Laser capture microdissection-reduced representation bisulfite sequencing and reduced representation bisulfite sequencing on human blood leukocyte, human endometrial tumor, mouse liver tissue, and mouse normal and neoplastic adrenal tissue
2013-03-21 | E-GEOD-45361 | biostudies-arrayexpress