The genome-wide methylation profile in peripheral blood was measured by an Illumina Infinium Human Methylation EPIC Beadchip covering over 850 000 CpG sites genome-wide. After quality control, normalization, and correction for batch and cell type, 275 samples with 800 587 DNAm probes remained. A threshold of false discovery rate-corrected P (PFDR) < 0.05 was applied to obtain URS-associated DNAm probes, leaving 30 538 probes that were enriched in promoter regulatory regions.
Infinium human methylationepic beadchip
Manufactured by Illumina
Sourced in United States, Canada
The Infinium Human MethylationEPIC BeadChip is a high-throughput DNA methylation microarray platform developed by Illumina. It enables the simultaneous measurement of the methylation status of over 850,000 CpG sites across the human genome.
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Lab products found in correlation
Ez 96 dna methylation kit, by Zymo Research (6 mentions)
Ez dna methylation kit, by Zymo Research (6 mentions)
Epitect plus bisulfite kit, by Qiagen (4 mentions)
Infinium humanmethylation450k beadchip, by Illumina (3 mentions)
Iscan system, by Illumina (3 mentions)
Zymo ez dna methylation kit, by Zymo Research (2 mentions)
Oragene kit, by DNA Genotek (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Ez dna methylation gold kit, by Zymo Research (2 mentions)
Genomestudio software, by Illumina (2 mentions)
Genomestudio v2011, by Illumina (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Dneasy kit, by Qiagen (1 mentions)
Paxgene blood dna kit, by Qiagen (1 mentions)
Genomestudio software v2011, by Illumina (1 mentions)
Qiamp mini kit, by Qiagen (1 mentions)
Infinium humanmethylation450 beadchip assay, by Illumina (1 mentions)
Genomestudio software version 2011, by Illumina (1 mentions)
Nanodrop nd 1000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Picogreen assay, by Thermo Fisher Scientific (1 mentions)
51 protocols using infinium human methylationepic beadchip
1
Genome-Wide DNA Methylation Analysis
2
ELITE Methylation Profiling for HT
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To maximize the likelihood of identifying associations between CpG sites and HT as a function of time-since-menopause, we selected subset of 48 women from the early/HT with the lowest rate of CIMT progression over the course of the trial for methylation profiling and an equivalent number of women from each of the early/placebo, late/placebo, and late/HT groups with the highest CIMT progression. Genomic DNA was extracted from buffy coats of this subset of 192 ELITE participants obtained at baseline and 36 months following randomization using DNeasy kits (Qiagen, Valencia, CA) and bisulfite treated with the Zymo EZ DNA Methylation Kit (Zymo Research, Orange, CA). Quantitative levels of DNA methylation were obtained for > 850,000 CpG sites using the Infinium Human Methylation EPIC BeadChip (Illumina, San Diego, CA) according to the manufacturer’s protocols.
3
Genome-wide DNA Methylation Profiling
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Genomic DNA (500 ng) was extracted from saliva samples using Oragene kits (DNA Genotek, Ottawa, Ontario, Canada) and treated with bisulfite reagents included in the EZ-96 DNA methylation kit (Zymo Research, Orange, CA, USA) following the manufacturer’s standard protocol. Bisulfite-converted DNA samples were then assessed with the Illumina Infinium Human MethylationEPIC BeadChip (Illumina, San Diego, CA, USA), which interrogates DNA methylation of >850,000 loci across the genome at single-nucleotide resolution.
4
Methylome Profiling of Peripheral Blood
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Genomic DNA was extracted from peripheral blood and bisulfite converted using the EpiTect PLUS Bisulfite Kit (QIAGEN, Germany). Sodium bisulfite converted DNA was then hybridized to the Illumina Infinium Human Methylation EPIC BeadChip to interrogate more than 850 000 CpG sites in the human genome at The Center for Applied Genomics (TCAG), Hospital for Sick Children Research Institute, Toronto, Ontario, Canada. Samples were run in two batches. On each microarray chip, cases and controls were randomly assigned a chip position. The minfi Bioconductor package in R was used to preprocess data including quality control, Illumina normalization and background subtraction, followed by extraction of beta (β) values as previously described (48 (link)). Standard quality control metrics in minfi were used, including median intensity QC plots, density plots and control probe plots. Probes with detection flaws (n = 816), probes near SNPs with minor allele frequencies above 1% (n = 29 958), cross-reactive probes (n = 41 975) (49 (link)), probes with raw beta of 0 or 1 in >0.25% of samples (n = 247), non-CpG probes (n = 2925) and X and Y chromosome probes (n = 19 627) were removed, resulting in a total of n = 774 245 probes remained for differential methylation analysis.
5
Infinium HumanMethylation EPIC BeadChip Protocol
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The 450 k DNAm data from the samples in our original study are described elsewhere [21 (link)]. The samples assessed on the Infinium HumanMethylation EPIC BeadChip (Illumina) were processed similar to the 450 k data set [21 (link)]. Samples were randomly allocated to sample plates and beadchips, as previously described [21 (link)].
6
Genome-wide DNA Methylation Analysis
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Genomic DNA (500 ng) was extracted from whole blood using PAXgene Blood DNA kits (Qiagen, Valencia, CA, USA) and standard procedures. Genomic DNA (500 ng) was treated with bisulfite reagents using a EZ-96 DNA methylation kit (Zymo Research, Orange, CA, USA) according to the manufacturer’s protocol. Bisulfite-converted DNA samples were used in the array-based genome-wide DNA methylation assay. Methylation status was assessed using the Illumina Infinium Human MethylationEPIC BeadChip (Illumina, San Diego, CA, USA), which interrogates DNA methylation >800,000 loci across the genome at single-nucleotide resolution.
7
Methylation Profiling with Infinium EPIC BeadChip
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Bisulfite-converted DNA samples from step 1 were assessed using Infinium Human Methylation EPIC Bead Chip (Illumina, California, USA) according to the manufacturer’s instructions. Illumina Genome Studio software V.2011.1 was used to extract the raw signal intensities of each probe (without background correction or normalization). The resulting raw data were normalized (control normalization) and background corrected by the manufacturer software to generate β-values for representing methylation status of each CpG site. The data preprocessing of MethylationEPIC BeadChip was performed by R package “ChAMP” V.2.18.2, including filter and normalization.22 (link) It filtered out (1) probes with detection p value (>0.01), (2) probes with <3 beads in at least 5% of samples per probe, (3) all non-CpG probes, (4) all SNP-related probes,23 24 (link) (5) all multihit probes,25 (link) (6) cross-reactive probes cohybridizing to the sex chromosomes.23 (link) MethylationEPIC BeadChip normalization (sample normalization, using the parameter “BMIQ”), calling differentially methylated points and calling DMRs (using the parameter “Bumphunter”) were performed.
8
Comprehensive DNA Methylation Analysis
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Genomic DNA was extracted in triplicate from early and Sen BMSCs and HUVECs and from BMSC-derived adipocytes and osteoblasts using Qiagen’s QiAmp mini kit following the manufacturer’s recommendations. Next, 1 μg DNA was bisulfite-converted using EZ DNA Methylation (Zymo Research, Irvine, CA, USA) and analyzed by the Infinium Human MethylationEPIC BeadChip (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s instructions. Raw data were extracted using minfi R package [20 (link)] and normalized using the preprocessFunnorm function. Not-reliable probes according to Zhou and collaborators [21 (link)] and probes on X and Y chromosomes were removed. The remaining 748,241 probes were annotated according to IlluminaHumanMethylationEPICanno.ilm10b4.hg19 annotation data.
9
Infinium Human MethylationEPIC BeadChip Analysis
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The Infinium Human MethylationEPIC BeadChip (Illumina) platform was used for DNA Methylation profiling. Samples were randomized on 2 slides and processed by the core facility for Bioinformatics and Expression Analysis (BEA), Karolinska Institutet, Huddinge campus. Idat files were loaded into R using scripts adapted from the ChAMP package (version 2.6.0) [13 (link)] referred to as “mEPIC.loading.Script.R” and “Champ.load.mEPIC.Script.R” available in Additional files 31 and 32 , respectively. Furthermore, an R script for normalization of mEPIC probes with Subset-quantile within array normalization (SWAN) [16 (link)] and BMIQ [21 (link)], referred to as “Champ.norm.mEPIC.Script.R” is available in Additional file 33 . Hierarchical clustering was performed with the ward.D2 method of the hclust function in R.
10
Comprehensive Epigenomic Analysis of Brain Tissue
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Extraction of DNA, sample randomization, epigenome-wide DNAm analysis, and quality control of methylation data was performed as described in Zillich et al. [13 (link)]. In brief, epigenome-wide DNAm was determined using the Infinium Human Methylation EPIC BeadChip (Illumina, San Diego, CA, USA) and quality control was performed with a customized and updated version of the CPACOR pipeline [14 (link)]. Samples from the NSWTRC were processed separately for each brain region in either a single or in two batches (ACC and PUT: one batch, BA9, CN and VS: two batches). The UTHealth samples were processed in a single batch after randomization according to sex, AUD status, and tissue. Methylation data were quantile-normalized.
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