Assessment of streptavidin bead binding capacity to improve quality of streptavidin-based enrichment studies
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Exploris 480
ORGANISM(S): Homo
SUBMITTER: 
Rebekah Gundry
PROVIDER: MSV000086213 | MassIVE |
REPOSITORIES: MassIVE
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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 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
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