Infinium hd methylation assay protocol

DNA methylation was analyzed in DNA extracted from fat cells using the Infinium Human Methylation 450 BeadChip assay (Illumina, San Diego, CA, USA). Genomic DNA (500 ng) was bisulfite treated using the EZ DNA methylation kit (Zymo Research, Orange, CA, USA) with the alternative incubation conditions recommended when using the Infinium Methylation Assay. The methylation assay was performed on 4 μl bisulfite-converted genomic DNA at 50 ng/μl according to the Infinium HD Methylation Assay protocol (Part #15019519, Illumina).
For validation of DMS in the independent cohort, bisulphite converted DNA from WAT specimens was hybridized to the Illumina Infinium 27K Human Methylation Beadchip v1.2 using standardized protocols (Illumina). DNA methylation data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo) and are accessible using GEO series accession numbers GSE67024 and GSE24884, respectively. The methylation assays were done at BEA (www.bea.ki.se).

DNA was isolated from PPAT using Puregene hisalt extraction method (Qiagen/Gentra). Briefly, the tissue was minced with scalpels in a sterile petri dish on ice and then transferred to Puregene Cell Kit for overnight Proteinase K digest at 55 °C. A second Proteinase K digest was done the next morning for 5 h. DNA from the digested tissue was purified using Puregene extraction protocol (Qiagen/Gentra). Purified DNA was washed 2× with 70% ethanol and DNA pellet air dried and rehydrated in TE (10 mM Tris-Cl, 1 mM EDTA pH 7.5). Epigenome-wide DNA methylation was analyzed using the Infinium Human Methylation450 (HM450) BeadChip (Illumina, San Diego, CA, USA) in the Center for Applied Genomics (Toronto). This array contains 485,577 probes, which cover 21,231 (99%) RefSeq genes. Briefly, DNA was bisulfite-converted using the EZ DNA methylation kit (Zymo Research, Orange, CA, USA) and then used on the Infinium Assay® followed by the Infinium HD Assay Methylation Protocol (Illumina). The imaging data on the BeadChips was captured by Illumina iScan system.

DNA was purified using QIAamp DNA Blood Mini Kit (QIAGEN), examined for integrity by agarose gel electrophoresis and quantified using Qubit 2.0 fluorimeter using a double stranded DNA (BR) assay (Thermo Fisher Scientific). About 500 ng of the sampled DNA was analyzed on a Infinium^® MethylationEPIC BeadChip (Illumina, San Diego, CA). The analysis comprised bisulfite conversion of DNA with EZ DNA Methylation^™ Kit (Zymo Research) using modified thermal conditions (as recommended by the supplier). Purified converted DNA was then used as an input to Infinium HD Assay Methylation Protocol (Illumina). The hybridized and stained arrays were ultimately scanned using HiScanSQ system (Illumina). The Infinium MethylationEPIC BeadChip used enabled the analysis of more than 850,000 methylation sites per sample which cover the broad content categories, including sites: within known CpG islands, outside of CpG islands, Non-CpG methylated sites identified in human stem cells (CHH sites), differentially methylated (DM) sites identified in tumor versus normal (multiple forms of cancer) and across several tissue types, FANTOM5 enhancers, ENCODE open chromatin and enhancers, DNase hypersensitivity sites and miRNA promoter regions. Moreover, the array content covers >90% of content contained on the previous Illumina HumanMethylation450 BeadChip.