## Description

These tracks display the methylation status of specific CpG dinucleotides in the given cell types as identified by the Illumina Infinium HumanMethylation27 BeadArray platform. In general, methylation of CpG sites within a promoter causes silencing of the gene associated with that promoter.

This method was used to validate Methyl-seq data generated in collaboration with the laboratory of Dr. Julie Baker at Stanford University to study methylation and gene expression changes that occur in human embryonic stem cells before and after differentiation to definitive endoderm [1]. Based on the results of these experiments, cut-off beta values to call "methylated" and "unmethylated" are selected.

Analysis is performed for two replicates for each cell line. Detailed information for the CpG targets is in an XLS formatted spreadsheet on the Myers' lab protocols website.

## Display Conventions

Scores associated with each site are beta value multiplied by 1000. Methylation status is color-coded as:

- orange = methylated (score >= 600)
- purple = partially methylated (200 < score < 600)
- bright blue = unmethylated (0 < score <= 200)
- black = NA (score = 0)

## Methods

Cells were grown according to the approved ENCODE cell culture protocols.

Genomic DNA was isolated from biological replicates of each cell line by using 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 UV absorbance.

The Methyl27K platform uses bisulfite treated genomic DNA to assay the methylation status of 27,578 CpG sites within more than 14,000 genes. Genomic DNA treated with sodium bisulfite converts unmethylated cytosine of a CpG dinucleotides into uracil; 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 MethylationTM 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.

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".

## Credits

Dr. Richard M. Myers
Mr. Yuya Kobayashi: yuyak@stanford. edu
Dr. Devin M. Absher: dabsher@hudsonalpha. org
Dr. Rebekka O. Sprouse: rsprouse@hudsonalpha. org

Contact: Flo Pauli.

## References

1. Brunner AL, Johnson DS, Kim SW, Valouev A, Reddy TE, Neff NF, Anton E, Medina C, Nguyen L, Chiao E et al. Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver. Genome Research. 2009 Jun;19(6):1044-56.

## Data Release Policy

Data users may freely use ENCODE data, but may not, without prior consent, submit publications that use an unpublished ENCODE dataset until nine months following the release of the dataset. This date is listed in the Restricted Until column on the track configuration page and the download page. The full data release policy for ENCODE is available here.