Genomestudio methylation module software

Signal measurement intensities were scanned in the 450k array using the Illumina iScanSQ platform. The intensity of the images was extracted with the GenomeStudio Methylation Software Module (v 1.9.0, Illumina). Methylation raw data are available in NCBI’s gene expression omnibus [65 (link)] as part of the MENA study through GEO series accession number GSE115278 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE115278).
β-Values were computed using the formula β-Value = M/(U + M) where M and U are the raw “methylated” and “unmethylated” signals, respectively. β-Values were corrected for type I and type II bias using the peak-based correction. Data were normalized in R using a categorical subset quantile normalization method (SQN) and probes associated with X and Y chromosomes were filtered out using the pipeline developed by Touleimat and Tost [66 (link)]. Probes with single nucleotide polymorphisms (SNPs) were also filtered out. Differences in methylation resulting from differences in cellular heterogeneity were corrected using estimateCellCounts function from minfi package for R statistical software [67 ], based on the Houseman method [68 (link)].

The bisulfite conversion of genomic DNA was carried out using EZ DNA Methylation Gold kit (Zymo Research). 200 ng converted DNA was analyzed using Illumina Infinium 450K Methylation array according to manufacturer's suggested protocols (Illumina). The quantitative value of DNA methylation was calculated from the ratio of fluorescent signals from the methylated alleles to the sum of the signals from the methylated and unmethylated alleles, assigned via Genome Studio Methylation Software Module (Illumina). Minfi package in Bioconductor (http://bioconductor.org/packages/release/bioc/html/minfi.html) was used for quality control and normalization before statistical analysis. Student t-test analysis from Limma package was used to identify differentially methylated genes with statistical significance between ND and CLL CD8⁺ T cells in methylation array data. A p-value < 0.05 and methylation difference > 0.25 were the cut off values used to select differentially methylated CpG sites.

DNA was extracted from PBMC, and the separated cell populations from the healthy adults and from whole blood in the Swedish search participants. For each sample, 500 ng of genomic DNA was bisulfite converted with the EZ-96 DNA Methylation Kit (Zymo Research Corporation, USA) according to the manufacturer’s instructions. Array-based-specific DNA methylation analysis was performed with the Infinium Human Methylation 450 K bead chip technology (Illumina, USA) as previously described [4 (link), 5 (link)]. Briefly, bisulfite-treated genomic DNA was whole-genome amplified, hybridized to HumanMethylation450 BeadChips (Illumina) and scanned using the Illumina iScan at the Mutation Analysis Core Facility (MAF) or Bioinformatics and Expression Analysis Core Facility (BEA) at Karolinska Institutet. The intensity of the images was extracted with the GenomeStudio Methylation Software Module (v 1.9.0, Illumina).

Genomic DNA from human neuroblastoma SH‐SY5Y cells stably expressing human AR containing 24 or 97 glutamine residues (AR‐24Q or AR‐97Q) was isolated from cell pellets using a DNA isolation kit for cells and tissues (Roche, Mannheim, Germany) according to the manufacturer's instructions. DNA concentrations were measured by spectrophotometry (NanoDrop ND‐1000 Spectrometer, Thermo Fisher Scientific, Wilmington, DE, USA), and DNA quality was estimated with the A260/A280 absorbance ratio. Array‐based specific DNA methylation analysis was performed using Human Methylation 450K BeadChip technology (Illumina, CA, USA). In this study, six samples were analyzed for more than 450,000 CpG sites at single nucleotide resolution with 96% coverage of CpG islands and 99% coverage of the RefSeq gene. Probes were distributed in CpG island shelves, CpG island shores, CpG islands, 5′ UTR promoter regions, first exons, gene bodies, and 3′ UTRs. The GenomeStudio Methylation Software Module (Illumina) was used to perform these analyses and assess image intensities.

The Illumina MethylationEPIC BeadChip is a comprehensive array that covers over 850,000 methylation sites quantitatively across the genome at single‐nucleotide resolution. It is a PCR‐free protocol that requires 250–1000 ng DNA input using the Infinium HD Assay. Genomic DNA is quantified by Qubit Fluorometer (Invitrogen) and qualified by agarose gel. Only DNA of high quality was selected and normalized to 25 ng/uL. Following a three step reaction, samples are bisulfite converted using the Zymo Research EZ DNA methylation kit. Once bisulfite conversion was complete, the Infinium HD protocol is followed. First, the DNA is amplified, fragmented, and precipitated to prepare for hybridization to the Beadchip. Twelve samples can be loaded onto one Beadchip. Single‐base extension of the oligos on the BeadChip is performed using the bisulfite‐converted DNA as a template. Beadchips are then scanned on the Illumina iScan or NextSeq systems. All raw data, along with corresponding manifest file, decode file, and sample sheet are uploaded into Illumina's GenomeStudio Methylation software module. Internal controls are checked within the software to validate complete bisulfite conversion and successful processing of the Infinium HD assay for each sample. Data are then ready for downstream analysis.