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Series GSE27584 Query DataSets for GSE27584
Status Public on Mar 01, 2011
Title DNA Methylation by Reduced Representation Bisulfite Seq from ENCODE/HudsonAlpha
Project ENCODE
Organism Homo sapiens
Experiment type Methylation profiling by high throughput sequencing
Summary This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Florencia Pauli If you have questions about the Genome Browser track associated with this data, contact ENCODE (

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 and
Overall design 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.
Web link
Contributor(s) Varley K, Gertz J, Myers RM
Citation(s) 22955980
BioProject PRJNA63443
Submission date Mar 01, 2011
Last update date May 15, 2019
Contact name ENCODE DCC
Organization name ENCODE DCC
Street address 300 Pasteur Dr
City Stanford
State/province CA
ZIP/Postal code 94305-5120
Country USA
Platforms (2)
GPL9052 Illumina Genome Analyzer (Homo sapiens)
GPL10999 Illumina Genome Analyzer IIx (Homo sapiens)
Samples (202)
GSM683759 Myers_BC_Leukocyte_UHN00204_2_None_BioChain
GSM683760 Myers_PrEC_2_None_UW
GSM683761 Myers_BC_Breast_02-03015_2_None_BioChain
SRA SRP006720

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE27584_RAW.tar 3.1 Gb (http)(custom) TAR (of BED, BEDRRBS)
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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