Unknown,Transcriptomics,Genomics,Proteomics

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CpG Methylation by Methyl 450K Bead Arrays from ENCODE/HAIB

ABSTRACT: 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 displays the methylation status of specific CpG dinucleotides in the given cell types as identified by the Illumina Infinium Human Methylation 450 Bead Array platform (http://www.illumina.com/products/methylation_450_beadchip_kits.ilmn). In general, methylation of CpG sites within a promoter causes silencing of the gene associated with that promoter. The Infinium Human Methylation 450 platform uses bisulfite treated genomic DNA to assay the methylation status of more than 450,000 CpG sites covering all designatable RefSeq genes, including promoter, 5' and 3' regions, without bias against those lacking CpG islands. Additionally, the assay includes CpG islands and shores, CpG sites outside of CpG islands, non-CpG methylated sites identified in human stem cells, differentially methylated sites identified in tumor versus normal (multiple forms of cancer) and across several tissue types, CpG islands outside of coding regions, miRNA promoter regions, and disease-associated regions identified through GWAS. Detailed information for the CpG targets is in an CSV formatted spreadsheet in the supplemental directory (http://hgdownload-test.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeHaibMethyl450/supplemental/). For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell). Genomic DNA was isolated from each cell line with the QIAGEN DNeasy Blood & Tissue Kit according to the instructions provided by the manufacturer. DNA concentrations and a level of quality of each preparation was determined by fluorescence with the Qubit Fluorometer (Invitrogen). Genomic DNA was treated with sodium bisulfite, converting unmethylated cytosines of CpG dinucleotides into uracils; methylated cytosines did not get converted. After bisulfite treatment, the methylation status of a site was assayed by single base-pair extension with a Cy3 or Cy5 labeled nucleotide on oligo-beads specific for the methylated or unmethylated state. The bisulfite conversion reaction was done using the Zymo Research EZ-96 DNA Methylation Kit (http://www.zymoresearch.com/product/ez-96-dna-methylation-kit-d5003). One step of the protocol was modified. During the incubation, a 30 second 95oC denaturing step every hour was included to increase reaction efficiency as recommended by the Illumina Infinium Human Methylation27 protocol. The bead arrays were run according to the protocol provided by Illumina (http://hgdownload-test.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeHaibMethyl450/supplemental/wgEncodeHaibMethyl450IlluminaProtocol.pdf). A beta value was calculated for each CpG target with Illumina's Bead Studio software with the Methylation Module v3.2. Beta-value = intensity value from the methylated bead type/(intensity values from the methylated + intensity value from unmethylated bead types + 100). The data was then quality-filtered using p-values. Beta values with p-value greater than 0.01 are considered to fall below the minimum intensity and threshold are displayed as "NA". Any beta value equal to or greater than 0.6 was considered fully methylated. Any beta value equal to or less than 0.2 was considered to be fully unmethylated. Beta values between 0.2 and 0.6 were considered to be partially methylated. Score in the bed files is beta value x 1000

ORGANISM(S): Homo sapiens

SUBMITTER: UCSC ENCODE DCC

PROVIDER: E-GEOD-40699 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 displays the methylation status of specific CpG dinucleotides in the given cell types as identified by the Illumina Infinium HumanMethylation27 BeadArray platform (http://www.illumina.com/pages.ilmn?ID=243). In general, methylation of CpG sites within a promoter causes silencing of the gene associated with that promoter. Detailed information for the CpG targets is in an XLS formatted spreadsheet on the Myers' lab protocols website (http://hudsonalpha.org/myers-lab/protocols). For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell). Genomic DNA was isolated from each cell line with the QIAGEN DNeasy Blood & Tissue Kit according to the instructions provided by the manufacturer. DNA concentrations and a level of quality of each preparation was determined by fluorescence with the Qubit Fluorometer (Invitrogen). The Methyl27K platform uses bisulfite treated genomic DNA to assay the methylation status of 27,578 CpG sites within more than 14,000 genes. When genomic DNA is treated with sodium bisulfite, unmethylated cytosine of CpG dinucleotides are converted into uracils; methylated cytosines do not get converted. After bisulfite treatment, the methylation status of a site is assayed by single base-pair extension with a Cy3 or Cy5 labeled nucleotide on oligo-beads specific for the methylated or unmethylated state. A beta value is calculated by Illumina's Bead Studio software for each CpG target. This value represents the intensity value from the methylated bead type divided by the sum of the intensity values from the methylated and unmethylated bead types for any given CpG target. Bisulfite conversion reaction was done using the Zymo Research EZ-96 DNA Methylation Kit (http://www.zymoresearch.com/epigenetics/dna-methylation/ez-96-dna-methylation-kit). One step of the protocol was modified. During the incubation, a 30 sec 95oC denaturing step every hour was included to increase reaction efficiency as recommended by the Illumina Infinium Human Methylation27 protocol. The bead arrays were run according to the protocol provided by Illumina (http://www.illumina.com/pagesnrn.ilmn?ID=275). The intensity data from the BeadArray was processed using Illumina's BeadStudio software with the Methylation Module v3.2. The data was then quality-filtered using p-values. Any beta value equal to or greater than 0.6 is considered fully methylated. Any beta value equal to or less than 0.2 is considered to be fully unmethylated. Beta values between 0.2 and 0.6 are considered to be partially methylated. Beta-values are quality filtered and spots that fall below the minimum intensity threshold are displayed as "NA". Score in the bed files is beta value x 1000

Project description:In the current study, we have performed a high-throughput CpG methylation analysis of well characterized and defined populations of human adipose-derived stem cells (hASCs) before and after in vitro induction of osteogenic and myogenic differentiation that allows identifying DNA methylation- regulated differentiation genes. We have also address the extent of the epigenetic programming of hASCs- derived differentiated cells by comparing the methylation profiling of these cells with their somatic counterparts from primary tissues. Finally, we also compared the patterns of CpG methylation of hASCs (and their derivatives) with the methylation profiles of myosarcoma and osteosrcoma cell lines. All the CpG methylation studies have been performed with the Infinium 27K methylation arrays (from Illumina). DNA from adipose M-bM-^@M-^Sderived stem cells (n=4), in vitro induced myocytes (n=3), in vitro induced osteocytes (n=3), primary osteocytes obtained from ribs (n=1), primary myocytes (n=1), osteosarcoma cell line (MG63) and myosarcoma cell lines (Te 32.T and RD) was isolated applying the QIAampM-BM-. DNA Mini Kit (Qiagen Iberia, Spain). Microarray- based DNA methylation profiling was performed with the HumanMethylation27 BeadChip Infinium Methylation ArraysM-BM-. (Illumina, Inc.).The panel was designed to compare the DNA methylation status of each group of samples, which allow interrogating 27,578 CpG loci covering 14,495 genes at single-nucleotide resolution by typing bisulfite-converted DNA. The sequences included in the panel are derived from the well-annotated NCBI CCDS database (Genome Build 36) and is supplemented with more than 1,000 cancer-related genes described in published literature. Probe content has been enriched to deeply cover more than 150 well-established cancer genes known to show differential methylation patterns. Methylation array content also targets the promoter regions of 110 miRNA genes. Methylation arrays were performed as follows. Briefly, bisulfite conversion of 1 M-NM-<g of genomic DNA was done using the CpGenomicTM DNA Modification Kit (Intergen Company, Purchase, NY, USA). After sodium bisulfite treatment, the remaining assay steps used Infinium technology and using Illumina-supplied reagents and conditions. A thermocycling program with a short denaturation step included for bisulfite conversion (16 cycles of 95M-BM-:C for 30 seconds followed by 50M-BM-:C for 1 hour) was performed to improve bisulfite conversion efficiency. After bisulfite conversion, each sample was whole-genome amplified (WGA) and enzymatically fragmented. The bisulfite-converted WGA-DNA samples were purified and applied to the BeadChips. During hybridization, the WGA-DNA molecules anneal to locus-specific DNA oligomers linked to individual bead types. The two bead types correspond to each CpG locus -one to the methylated and the other to the unmethylated state. Allele-specific primer annealing is followed by single-base extension using DNP- and Biotin-labeled dNTPs. Both bead types for the same CpG locus will incorporate the same type of labelled nucleotide, determined by the base preceding the interrogated cytosine in the CpG locus, and therefore will be detected in the same colour channel. After extension, the array is fluorescently stained, scanned, and the intensities of the unmethylated and methylated bead types measured. DNA methylation values, described as beta values, are recorded for each locus in each sample via BeadStudio software. DNA methylation beta values are continuous variables between 0 (completely unmethylated) and 1 (completely methylated), representing the ratio of the intensity of the methylated bead type to the combined locus intensity.

Project description:Methylation of DNA at CpG dinucleotides represents one of the most important epigenetic mechanisms involved in the control of gene expression in vertebrate cells. In this report, we conducted high-throughput nucleosome reconstitution experiments on 572 KB of human DNA and 668 KB of mouse DNA that was unmethylated or methylated in order to investigate the effects of this epigenetic modification on the positioning and stability of nucleosomes. The DNA loci from both species contained the genes that encode the serum albumin family, the MYC protein, and the tumor suppressor p53. The results demonstrated that a small subset of nucleosomes positioned by nucleotide sequence was sensitive to methylation where the modification increased the affinity of these sequences for the histone octamer. The features that distinguished these nucleosomes from the bulk of the methylation-insensitive nucleosomes were an increase in the frequency of CpG dinucleotides and a unique rotational orientation of CpGs such that their minor grooves tended to face toward the histones in the nucleosome rather than away. We propose that these features serve to enhance the affinity of methylated DNA for the histone octamer and that this effect could be involved in gene regulatory mechanisms such as silencing. These methylation-sensitive nucleosomes were preferentially associated with exons as compared to introns while unmethylated CpG islands near transcription start sites became enriched in nucleosomes upon methylation. The results of this study suggest that the effects of DNA methylation on nucleosome stability in vitro can recapitulate what has been observed in the cell and provide a direct link between DNA methylation and the structure and function of chromatin. For the human data, there were 3 unmethylated nucleosome replicates, 2 methylated nucleosome replicates, an unmethylated MNase control, and an unmethylated sonicated control. For the mouse data, there were 2 unmethylated nucleosome replicates, 2 methylated nucleosome replicates, an unmethylated MNase control, a methylated MNase control, and an unmethylated sonicated control.

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CpG Methylation by Methyl 450K Bead Arrays from ENCODE/HAIB

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