Infinium methylationepic beadchip array

Manufactured by Illumina

Sourced in United States, Germany, United Kingdom

The Infinium MethylationEPIC BeadChip array is a microarray-based technology designed to measure DNA methylation levels across the human genome. It is capable of analyzing over 850,000 methylation sites, covering 99% of RefSeq genes, with a focus on CpG islands and regulatory regions.

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85 protocols using infinium methylationepic beadchip array

Profiling EWSR1-ETS-Dependent CpG Methylation

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For analysis of EWSR1-ETS-dependent CpG island methylation, genomic DNA of all 18 EwS cell lines with and without knockdown of the respective fusion oncogene was genotyped on Illumina Infinium MethylationEPIC BeadChip arrays. Therefore, all EwS cell lines were seeded in three technical replicates in standard medium or standard medium supplemented with dox (1 μg/mL). Medium was changed to fresh medium after 48 h (including dox). After 96 h, samples were lysed and DNA was extracted with the NucleoSpin Tissue kit (Macherey Nagel). Genomic DNA was genotyped with the Illumina Infinium Methylation EPIC BeadChip arrays at the Molecular Epidemiology unit of the German Research Center for Environmental Health (Helmholtz Center, Munich, Germany). Readout and analysis of the EPIC arrays was performed with GenomeStudio (Illumina) and the R minfi package and bumphunter algorithm.^{98 (link)} For t-SNE analysis, the same approach and tools as for transcriptome and proteome data were applied. Methylation profile classification was run on the MolecularNeuropathology.org website.^{61 (link),100 (link)}

Yoshimura S., Takagi Y., Harada J., Teramoto T., Thomas S.S., Waeber C., Bakowska J.C., Breakefield X.O, & Moskowitz M.A. (2001). FGF-2 regulation of neurogenesis in adult hippocampus after brain injury. Proceedings of the National Academy of Sciences of the United States of America, 98(10), 5874-5879.

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Profiling EWSR1-ETS-Dependent CpG Methylation

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Orth M.F., Surdez D., Faehling T., Ehlers A.C., Marchetto A., Grossetête S., Volckmann R., Zwijnenburg D.A., Gerke J.S., Zaidi S., Alonso J., Sastre A., Baulande S., Sill M., Cidre-Aranaz F., Ohmura S., Kirchner T., Hauck S.M., Reischl E., Gymrek M., Pfister S.M., Strauch K., Koster J., Delattre O, & Grünewald T.G. (2022). Systematic multi-omics cell line profiling uncovers principles of Ewing sarcoma fusion oncogene-mediated gene regulation. Cell reports, 41(10), 111761.

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Epigenomic Profiling of Circulating cfDNA in Ovarian Cancer

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Circulating cfDNA (cFDNA) was profiled using Illumina Infinium MethylationEPIC BeadChip arrays (Illumina, San Diego, CA). In this prospective case–control study, we compared the methylation profile of OC patients with matched age grouped controls. The study was approved by the Institutional Review Board of Beaumont Health System, Royal Oak, MI, USA (IRB#2018-306) and performed in accordance with the Declaration of Helsinki. All participants provided written informed consent. CfDNA was extracted using the blood from 5 OC female cases and 12 female controls. Blood samples were collected in Streck Cell-Free DNA BCT^® tubes^{19 (link)}. Samples were deidentified and processed within 24 h of sample collection. For the primary processing, these were centrifuged at 3000×g for 15 min and the plasma was aliquoted into cryogenic vials. The aliquoted plasma samples were stored at − 80 °C until further process. Using QIAamp circulating nucleic acid kit (Qiagen Cat # 55114) the ccfDNA was extracted. All samples were bisulfite converted using EZ DNA Methylation Kit (Zymo, USA) per the manufacturer’s standardized method. Illumina Infinium MethylationEPIC BeadChip arrays (~ 850,000 CpG loci genome-wide) was used for methylation analysis based on manufacturer’s standardized protocol^{20 (link)}.

Bahado-Singh R.O., Ibrahim A., Al-Wahab Z., Aydas B., Radhakrishna U., Yilmaz A, & Vishweswaraiah S. (2022). Precision gynecologic oncology: circulating cell free DNA epigenomic analysis, artificial intelligence and the accurate detection of ovarian cancer. Scientific Reports, 12, 18625.

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Methylation Profiling of CD8+ T Cells

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Methylation status of CD8+ T cells was measured by hybridising bisulfite-converted DNA to the Infinium MethylationEPIC Bead-Chip array (Illumina). Methylation β values were calculated from raw intensity values using GenomeStudio Methylation module software (Illumina) after subtracting background, normalisation to control probes, error correction using the Illumina custom model and correction for false discovery rate. Group comparison and statistical significance were determined using

β = β_{S L E} - β_{Control} and D i f f S c o r e = 10 \times s g n (β_{S L E} - β_{Control}) \times \log_{10} (P),

respectively. Probes were filtered using the following criteria:

| Δ β | \geq 0.10, DiffScore | \geq 22

(equivalent to an FDR-adjusted p≤0.01), detection p<0.05 and no single nucleotide polymorphism (SNP) within 10 bp of the 3’ end of the probe. Gene annotations for hypermethylated and hypomethylated probes were used as input for gene function annotation analysis using DAVID.^{24 (link)} Categories were restricted to molecular function and biological process gene ontologies and KEGG pathways and only annotations with a minimum gene number of 2, a modified Fisher’s exact p<0.1 and FDR≤5% were considered. Raw and processed DNA methylation data from patients and controls have been deposited in Gene Expression Omnibus (GEO accession number GSE123003).

Miller S., Tsou P.S., Coit P., Gensterblum-Miller E., Renauer P., Rohraff D.M., Kilian N.C., Schonfeld M, & Sawalha A.H. (2019). Hypomethylation of STAT1 and HLA-DRB1 is associated with type-I interferon-dependent HLA-DRB1 expression in lupus CD8+ T cells. Annals of the rheumatic diseases, 78(4), 519-528.

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Preprocessing DNA Methylation Data

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Bisulfite-modified DNA samples were subjected to the Infinium™ MethylationEPIC BeadChip array (Illumina, Inc., CA, USA) and measured for their methylation intensity at >850,000 CpG sites. Probe intensity data (iDAT) files were processed via the preprocessNoob normalization method from the R package minfi [34 (link)]. ENmix [35 (link)], another R package, was performed for the quality control of probes. For sample selection, we set that samples with more than 5% of probes with a detection p-value > 1.0 × 10^-6 would be excluded to distinguish from background noise; fortunately, all samples had good quality and were kept in the study. Probes were checked by observing any probes that were not missing in more than 10% of the samples. BMIQ [36 (link)] from the watermelon [37 (link)] R package was performed for probe-type normalization, and batch effects were corrected using ComBat [38 (link)]. Then, 119,258 probes previously described to be potentially cross-hybridizing, sex-specific, non-CpG methylation and SNP-associated were filtered [39 (link)]. In total, 746,980 were included in the final analysis (Figure 1A). The CpG loci were annotated with the IlluminaHumanMethylationEPICanno.ilm10b4.hg19 [40 ] R package.

Chen J.Q., Salas L.A., Wiencke J.K., Koestler D.C., Molinaro A.M., Andrew A.S., Seigne J.D., Karagas M.R., Kelsey K.T, & Christensen B.C. (2024). Matched analysis of detailed peripheral blood and tumor immune microenvironment profiles in bladder cancer. Epigenomics, 16(1), 41-56.

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DNA Methylation Profiling using Infinium MethylationEPIC

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Peripheral blood was collected from enrolled participants into sodium citrate tubes. DNA was isolated using the Chemagic™ Prime™ instrument, an automated chemical extraction machine that uses magnetized rods to separate nucleic acids from solutions. A Fragment Analyzer (Agilent) was used to measure DNA length, and the 260/280 optical density (OD) ratio was used to assess DNA purity. Samples with an OD 260/280 ratio of 1.6~2.0 were considered pure. DNA samples were subjected to sodium bisulfite treatment using the EZ DNA Methylation Kit (Zymo Research, CA, USA). DNA methylation at each cytosine-phosphate-guanine dinucleotide (CpG) site was quantified with the Infinium® MethylationEPIC BeadChip array (Illumina Inc) which covered 850,000 methylation sites [79 (link)]. Samples were randomized on the MethylationEPIC BeadChip to avoid batch effects. The assay was performed according to the manufacturer’s standard protocol.

Tseng C.C., Liao W.T., Wong M.C., Chen C.J., Lee S.C., Yen J.H, & Chang S.J. (2021). Cell lineage-specific methylome and genome alterations in gout. Aging (Albany NY), 13(3), 3843-3865.

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Illumina Infinium MethylationEPIC BeadChip Array Processing

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The raw IDAT files (two per sample) generated by Illumina Infinium MethylationEPIC BeadChip Array (850 K) were preprocessed by using the minfi (v1.25.1) R/Bioconductor package^{94 (link)}. Preprocessing steps included background correction, dye bias normalization, calculation of beta values and corresponding p value detection. Probes with a detection p value greater than 0.01 in a given sample were deemed not to be statistically significantly different from background and were thus excluded from the analyses. The following filtering criteria were applied: (1) removal of probes designed for sequences on X and Y chromosomes; (2) probes within promoter regions defined as (−1500, +1500) bp of the transcription start sites (TSSs); and (3) probes located in CpG islands.

Jiang Y., Wang J., Sun M., Zuo D., Wang H., Shen J., Jiang W., Mu H., Ma X., Yin F., Lin J., Wang C., Yu S., Jiang L., Lv G., Liu F., Xue L., Tian K., Wang G., Zhou Z., Lv Y., Wang Z., Zhang T., Xu J., Yang L., Zhao K., Sun W., Tang Y., Cai Z., Wang S, & Hua Y. (2022). Multi-omics analysis identifies osteosarcoma subtypes with distinct prognosis indicating stratified treatment. Nature Communications, 13, 7207.

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Genome-wide DNA Methylation Profiling

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We carried out bisulfite conversion of 600 ng of DNA extracted from cell lines using the EZ DNA Methylation kit (Zymo Research, Proteigene, Saint-Marcel, France). The genome-wide profiling of DNA methylome was determined using the Infinium MethylationEPIC BeadChip array (Illumina, Paris, France) at the Functional Genomics Facility of the INSERM unit UMR_S 1256 (NGERE, Vandoeuvre-lès-Nancy, France). The Infinium MethylationEPIC BeadChip provides coverage of > 850,000 CpG probes in enhancer regions, gene bodies, promoters, and CpG islands. The arrays were scanned on an Illumina iScan® system, and raw methylation data were extracted using Illumina’s Genome Studio methylation module. For each CpG probe, the methylation level was described as a β value, ranging between 0 (fully unmethylated CpG probe) and 1 (fully methylated CpG probe). Background correction and normalization were implemented using the SWAN method (R Package Minfi) [29 ]. Probe annotation information, including sequence and chromosome location for the Infinium MethylationEPIC BeadChip array, was retrieved from the Infinium MethylationEPIC v1.0 B5 Manifest File.

Siblini Y., Chéry C., Rouyer P., Raso J., Julien A., Hergalant S., François A., Bezdetnaya L., Vogin G., Guéant J.L, & Oussalah A. (2021). Ionizing radiations induce shared epigenomic signatures unraveling adaptive mechanisms of cancerous cell lines with or without methionine dependency. Clinical Epigenetics, 13, 212.

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Illumina Infinium Methylation EPIC Profiling

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One microgram of DNA was bisulfite-converted using an EZ DNA Methylation-Gold Kit (ZYMO), and the DNA was whole-genome amplified, enzymatically fragmented, purified, and applied to an Illumina Infinium Methylation EPIC BeadChip Array according to the Illumina methylation protocol. DNA methylation IDAT files were processed in R using the minfi package. Probes with fewer than three beads for either the methylated or unmethylated channel or with a detection P value ≥ 0.01 were removed. Probes with SNPs or their single base extension, X chromosome, or Y chromosome at the CpG site were excluded. After quality control filtering, 829,120 CpGs in 25 samples remained for later analysis. DNA methylation files were processed and normalized by R software packages using the ChAMP package. For each of the samples, CpG sites with a detection P value less than 0.05 were excluded from the analysis. In addition, probes with SNPs or their single base extension, X chromosome and Y chromosome at the CpG site were excluded. The standard DMSs were δ beta| > 0.1 and p < 0.05 (Wilcoxon test).

Liu Z., Li X., Gao Y., Liu J., Feng Y., Liu Y., Wang J., Wang C., Wang D., He J., Han W., Mei Q, & Sun Y. (2023). Epigenetic reprogramming of Runx3 reinforces CD8 + T-cell function and improves the clinical response to immunotherapy. Molecular Cancer, 22, 84.

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Quantifying DNA Methylation Across the Genome

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Before and after the 1-week intervention, saliva samples (1 ml) from all the participants (n = 22/group) were collected using Oragene saliva collection kits and DNA was isolated according to the manufacturer’s protocol. DNA concentration was determined using a Qubit fluorometer (Life Technologies) and normalized to 20 ng/μl for the methylation microarray. Bisulfite conversion was performed with the EZ methylation Gold-kit (cat# D5005, Zymo Research) and the Illumina Infinium MethylationEPIC Beadchip Array was used to quantitatively interrogate at single-nucleotide resolution over 850,000 CpG sites across the genome (Biotech Center, University of Wisconsin-Madison).

Kaliman P., Cosín-Tomás M., Madrid A., Roque López S., Llanez-Anaya E., Papale L.A., Alisch R.S, & Davidson R.J. (2022). Epigenetic impact of a 1-week intensive multimodal group program for adolescents with multiple adverse childhood experiences. Scientific Reports, 12, 17177.

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