DNA methylation was measured in peripheral whole blood samples from participants at age 17 y using the Illumina Infinium HumanMethylation450K BeadChip. Venous blood samples were taken by phlebotomists after an overnight fast. Samples were stored at (176°F) until analysis. Processing of the Illumina Infinium HumanMethylation450K BeadChips was carried out by the Centre for Molecular Medicine and Therapeutics (CMMT) (http://www.cmmt.ubc.ca ). We excluded three samples as outliers and one sample for biological sex inconsistency, because this might be indicative of a sample mix-up. Outliers were defined by the R packages shinyMethyl (version 1.22.0, Bioconductor) (Fortin and Hansen 2014 (link)) and MethylAid (version 1.22.0, Bioconductor) (Van Iterson et al. 2014 (link)) as samples that did not cluster together with the rest. Annotation of the CpG to the nearest gene was performed using Illumina’s genome coordinates (GRCh37/hg19). DNA methylation data of 996 white study participants on 475,429 probes were available for analysis.
Infinium humanmethylation450k beadchip
Manufactured by Illumina
Sourced in United States
The Infinium HumanMethylation450K BeadChip is a lab equipment product designed for genome-wide DNA methylation analysis. It targets over 485,000 methylation sites across the human genome, providing a comprehensive view of the methylome. The BeadChip is intended for use in epigenetic research applications.
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Lab products found in correlation
Ez dna methylation kit, by Zymo Research (24 mentions)
Genomestudio software, by Illumina (8 mentions)
Human methylation 450k beadchip, by Illumina (4 mentions)
Hiscan system, by Illumina (4 mentions)
Zymo ez 96 dna methylation kit, by Zymo Research (4 mentions)
Iscan system, by Illumina (3 mentions)
Ez 96 dna methylation kit, by Zymo Research (3 mentions)
Genome studio program methylation module, by Illumina (3 mentions)
Infinium human methylationepic beadchip, by Illumina (3 mentions)
Qiaamp dna mini kit, by Qiagen (3 mentions)
Epitect 96 bisulfite kit, by Qiagen (2 mentions)
Peripheral blood leukocytes, by Promega (2 mentions)
Infinium methylationepic beadchip, by Illumina (2 mentions)
Biorobot universal system, by Qiagen (2 mentions)
Qiaamp dna blood biorobot mdx kit, by Qiagen (2 mentions)
Iscan instrument, by Illumina (2 mentions)
Zymo ez dna methylation kit, by Zymo Research (2 mentions)
Qiaamp dna blood maxi kit, by Qiagen (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Human610 quad beadchip, by Illumina (1 mentions)
142 protocols using infinium humanmethylation450k beadchip
1
Epigenetic Analysis of Whole Blood DNA Methylation
2
Genome-wide DNA Methylation Profiling of hESCs
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Genomic DNA bisulfite conversion and hybridization to Infinium Human Methylation 450k Bead-Chip (Illumina, Inc., CA, USA) was performed at the Human Genotyping Unit of the Spanish National Cancer Center (CEGEN-CNIO, Madrid, Spain), as described previously [34] .
Data from the Infinium Human Methylation 450k BeadChip of four human embryonic stem cell (hESC) lines (hESC1-4) have been previously reported [35] .
Data from the Infinium Human Methylation 450k BeadChip of four human embryonic stem cell (hESC) lines (hESC1-4) have been previously reported [35] .
3
Infinium HumanMethylation450K Assay Protocol
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The Infinium HumanMethylation450K BeadChip (Illumina) includes 485,577 CpG sites located throughout the genome [36 (link)]. Genomic DNA from HCT116 cells [mock, irradiated (2 and 5 Gy), and 5-aza-dC (0.5 μM for 72 hrs) treated] was isolated using a phenol/chloroform method. Bisulfite modification of genomic DNA was performed using an EZ DNA Methylation Kit (Zymo Research). The bisulfite conversion efficiency was determined by sample-dependent controls on the chip and was displayed in the quality control panel in the software. All samples passed quality control measurements. The samples were run on an Infinium HumanMethylation450K BeadChip (Illumina) and scanned on an Illumina iScan instrument according to the manufacturer’s instructions. The methylation values for individual CpG sites in each sample were obtained as β-values. The β-value generated for each CpG locus measure the intensity of methylated (β = 1) and unmethylated probes (β = 0). The β-value is a continuous variable that is calculated by dividing the intensity of the methylated beads by the combined intensity, and these values range from 0 to 1. A CpG locus was considered differentially methylated if the β-value was > = |0.2| and its ratio was > |1.5|X fold compared to control (mock treated). This cut-off value of |0.2| represents the 99% confidence interval of the detection limit [13 (link)].
4
Copy Number and Methylation Analysis of S1 Tumors
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At the time of the original gene expression study [4 ], a pilot set of five S1 tumors with available tissue were analyzed for copy number and methylation analysis. Copy number analysis was performed using the Illumina Human 610-quad beadchip, as described in S1 File .
Methylation analysis was performed using the Illumina Infinium Human Methylation 450K BeadChips according to the manufacturer’s protocol using methods previously reported [14 (link)]. The average of the beta values for probes on 19q in the test set (S1 tumors) was compared with those of 11 CCSKs; regions were identified in which the average beta value for ≥ 5 consecutive probes ranged from 40–60% in the comparison set and ranged from 0–25% or from 75–100% in the test set. The regions were visualized with Integrative Genomics Viewer [60 (link), 61 (link)].
Methylation analysis was performed using the Illumina Infinium Human Methylation 450K BeadChips according to the manufacturer’s protocol using methods previously reported [14 (link)]. The average of the beta values for probes on 19q in the test set (S1 tumors) was compared with those of 11 CCSKs; regions were identified in which the average beta value for ≥ 5 consecutive probes ranged from 40–60% in the comparison set and ranged from 0–25% or from 75–100% in the test set. The regions were visualized with Integrative Genomics Viewer [60 (link), 61 (link)].
5
Methylation Analysis of Gene Expression
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Methylation analysis was performed on 11 samples for which sufficient DNA was available, using Illumina Infinium Human Methylation 450K BeadChips (Illumina, San Diego, CA, USA) according to the manufacturer's protocol [54 (link)]. ß-values were calculated from GenomeStudio v2010.1. Integrative genome viewer (IGV) was utilized to visualize methylation data (http://www.broadinstitute.org/igv/ ). Data were correlated with gene expression data as follows: methylation probes located in the gene body, or within 10k base pairs upstream or downstream of a gene were identified. For each gene, the expression was determined by using the probe with the highest expression. For each probe and gene pair, the correlation between methylation and gene expression was analyzed using the Generalized Linear Model (GLM), which is implemented in R (http://www.R-project.org/ ). P-values were adjusted for multiple comparisons using the multitest package in R. A negative t-value indicates that the methylation and expression levels are inversely correlated. A correlation with adjusted p-value < 0.05 was considered significant.
6
Comprehensive PNEN Methylation Analysis
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Infinium HumanMethylation450K BeadChips (Illumina) DNA methylation data of additional PNEN patients and alpha and beta cell samples were collected from public repositories (Figure 1 ). Neimann et al. isolated alpha and beta cell samples from, respectively, two and three donors and subjected the samples to DNA methylation analysis [17 (link)]. Furthermore, DNA methylation data were obtained from 32 PNENs analyzed by Chan et al. [15 (link)], 5 PNENs analyzed by Timp et al. [22 (link)] and 5 PNENs analyzed within the PAAD project of the TCGA Research Network (https://www.cancer.gov/tcga ). We obtained available clinicopathological data and included the mutation status of the ATRX, DAXX and MEN1 genes, if available.
7
DNA Methylation Profiling Using Illumina 450K
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DNA methylation was measured by using Illumina’s Infinium Human Methylation450K BeadChips (“450K bead array”) according to the manufacturer’s manual. DNA bisulfite conversion was carried out using EZ DNA Methylation Kit (Zymo Research) by following manufacturer’s manual with modifications for Illumina Infinium Methylation Assay. Briefly, 400 ng of genomic DNA was first mixed with 5 ul of M-Dilution Buffer and incubated at 37 °C for 15 min and then mixed with 100 ul of CT Conversion Reagent prepared as instructed in the kit’s manual. Mixtures were incubated in a thermocycler with 16 thermal cycles at 95 °C for 30 s and 50C for 1 hour. Bisulfite-converted DNA samples were loaded onto 96-column plates provided in the kit for desulphonation and purification. Concentration of eluted DNA was measured using Nanodrop-1000 spectrometer.
8
Illumina DNA Methylation Profiling Workflow
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DNA methylation was evaluated using the Illumina Infinium Human Methylation 450 K BeadChips (HM450K; Illumina)100 (link). Briefly, 1 μg of genomic DNA was bisulfite converted using the EZ DNA Methylation-Direct kit (Zymo Research Irvine, CA). The bisulfite conversion efficacy was evaluated using the MethyLight assay for a panel of defined markers. Samples passing quality control (QC) were whole genome amplified, enzymatically fragmented, hybridized onto HM450K BeadChips, and scanned using the Illumina iScan microarray scanner (Illumina). The raw data (in IDAT file format) was processed with the ‘lumi’ R package using default parameters, and DNA methylation values were calculated as M values for use in subsequent analyses. Annotation of HumanMethylation450 probes was downloaded from the Illumina website (‘https://support.illumina.com/downloads/humanmethylation450_15017482_v1-2_product_files.html ’). Probes associated with individual genes were corrected according to gene location information from GENCODE Human v19. Briefly, probes located within 2000 bp upstream of the gene start site were annotated as ‘TSS2000’ or ‘Promoter’, and probes located between gene start and gene end were annotated as ‘GeneBody’. Existing annotations of 5’UTR or 3’UTR were retained. DNA methylation data were not available for two cell lines in the refined cohort (S2350, S2667).
9
Genome-wide DNA Methylation Profiling of TET2 Mutations
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DNA was extracted from HEL cell clones using a DNA Mini Kit (QIAGEN) and sent for processing and hybridization to Infinium HumanMethylation450K BeadChips (Illumina) by Eurofins Genomics. Data processing and analysis were performed according to an established workflow (59 (link)). Specifically, raw intensity data files containing methylated (M) and unmethylated (U) intensity measurements were imported into R, and the minfi Bioconductor package (60 (link)) was used to calculate detection P values (detP), normalize data (using the preprocessFunnorm function), and generate β (β = M/[M+U+100]) and M (M = log2[M/U]) values for individual CpG probes. Poorly performing probes (detP < 0.01) and those interrogating SNPs were removed, leaving 410,811 probes in the final data set. The limma Bioconductor package (61 (link)) was used to identify significantly differentially methylated probes based on TET2 mutation status (monoallelic versus biallelic) using M values. Resulting P values were adjusted to control for FDR (5%) (58 ), and significantly differentially methylated CpGs were defined as those with FDR-adjusted P < 0.05 and |log2FC| ≥ 2. Unsupervised hierarchical clustering based on M values was performed in R with scaling by SD.
10
Genome-wide DNA Methylation Analysis
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Genomic DNA was bisulfite converted utilizing the EZ DNA Methylation kit (ZymoResearch, Irvine, CA, USA) following the manufacturer’s instructions. Converted DNA was further processed and hybridized to Infinium HumanMethylation 450 k BeadChips (Illumina Inc., San Diego, CA, USA) following the standard Illumina workflow. Hybridized chips were scanned with an Illumina iScan system on default settings. All chips passed the manufacturer’s standard quality controls as well as further quality controls applied within the downstream in silico analysis. Due to a technical error, three samples were lost during processing, limiting the total number of samples from which methylation data was available to 26 (9 asthma, 10 COPD, 7 controls).
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