Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track was produced as part of the ENCODE project. It reports the percentage of DNA molecules that exhibit cytosine methylation. 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 was assayed with Whole Genome Bisulfite Sequencing (WGBS). Genomic DNA was sheared by sonication, end-repaired and then ligated to methylated sequencing adapters. The library fragments were treated with sodium bisulfite and amplified by PCR to convert every unmethylated cytosine to a thymine while leaving methylated cytosines intact. The sequenced fragments were aligned to a bisulfite-converted reference genome. For each assayed cytosine, the number of sequencing reads covering that C and the percentage of those reads that were methylated were reported. 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 cytosines across the genome was assayed with Whole Genome Bisulfite Sequencing (WGBS). WGBS was performed on cell lines grown by ENCODE production groups. WGBS was carried out by the Myers production group at the HudsonAlpha Institute for Biotechnology. Isolation of Genomic DNA: Genomic DNA was isolated from 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 were determined using fluorescent DNA-binding dye and a fluorometer (Invitrogen Quant-iT dsDNA High Sensitivity Kit and Qubit Fluorometer). Typically, 2 µg of genomic DNA is used to make WGBS libraries. WGBS Library Construction and Sequencing: WGBS library construction started with sonication of genomic DNA on a Covaris S2 instrument. Sheared ends were then repaired and blunted with DNA polymerase I, T4 DNA polymerase and T4 polynucleotide kinase in the presence of dATP, dGTP and dTTP. After end repair, Klenow exo- DNA Polymerase was used to add an adenosine as a 3' overhang. Next, a methylated version of the Illumina paired-end adapters was ligated onto the DNA. Adapter-ligated 400 bp genomic DNA fragments were selected using a 2% agarose SizeSelect E-gel. The selected adapter-ligated fragments were 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 was amplified in a final PCR reaction which was optimized to uniformly amplify diverse fragment sizes and sequence contexts in the same reaction. During this final PCR reaction, uracils were copied as thymines, resulting in a thymine in the PCR products wherever an unmethylated cytosine existed in the genomic DNA. These libraries were then sequenced with an Illumina HiSeq 2000 according to the manufacturer's recommendations as paired-end 50 bp reads. Libraries were sequenced to a depth of 600 million aligned reads. Data Analysis: To analyze the sequence data, Bismark (Krueger and Andrews, 2011) was used to align sequences reads. Generally, each read went through a conversion of Cs to Ts and was then aligned to fully converted plus and minus strands of the hg19 build of the human genome. A few custom refinements were made to the Bismark program. Since these libraries were made in a directional orientation with the first read always being C-poor, we skipped unnecessary alignments to impossible orientations. We also implemented a more stringent uniqueness filter, only allowing reads that have one acceptable alignment (based on default Bowtie parameters) across both strands. Once reads were aligned, the percent methylation was calculated for each cytosine using the original sequence reads. The percent methylation and number of reads is reported for each CpG in the wgEncodeHaibMethylWgbsXXXXCpg.bigBed file and for each non CpG cytosine in the wgEncodeHaibMethylWgbsXXXXNoncpg.bigBed file.

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.

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: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

Project description:Cytosine methylation in the genome of Drosophila melanogaster has been elusive and controversial: methylcytosine has been detected at very low levels in early embryos, but the genomic location and function of methylation has not been established. We have mapped cytosine methylation genomewide in Stage 5 Drosophila embryo DNA by combining immuno-enrichment for 5-methylcytosine, bisulfite conversion, and deep sequencing. Unlike methylation patterns observed in other eukaryotic species, methylation in Drosophila is punctate and highly strand-asymmetrical; we confirmed this by direct PCR amplification and sequencing of bisulfite-converted DNA. Despite the locally asymmetric nature of methylation, large-scale patterns of methylation are symmetric. Methylated regions make up ~1% of the genome, and within these regions methylation of individual cytosines averages 2-10%. Methylation is concentrated in specific 5-base sequence motifs that are CA- and CT-rich but depleted of guanine. It is depleted from promoters, coding sequences, and most retrotransposons, and enriched in introns and in certain simple sequence repeats containing the commonly methylated motifs. Comparison with available gene expression data indicates that methylation in a gene is associated with lower expression; the X chromosome, which is subject to gene dosage compensation, is more densely methylated than the autosomes. This study firmly establishes the presence of cytosine methylation in Drosophila; the temporal overlap of methylation with the maternal-zygotic transition raises the possibility that methylation participates in the transition to zygotic gene expression. To enrich for rare cytosine methylation in Drosophila at embryonic Stage 5 (2-3 hours post-fertilization), we enriched sonicated Stage 5 genomic DNA for methylcytosine by immunoprecipitation with antibody to 5-methylcytosine. The immunoprecipitated DNA was then bisulfite converted and Illumina sequenced to obtain direct evidence for the presence of methylation. The presence and extent of DNA methylation was confirmed by Illumina sequencing of bisulfite-converted PCR amplicons.

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Whole Genome Bisulfite Sequencing by ENCODE/HAIB

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