Genome-Wide DNA Methylation Analysis in Liver Tissue
Genomic DNA (gDNA) was extracted from flash-frozen liver tissue with PureLink Genomic DNA Mini Kit (Thermo) and quantified using Nanodrop (Thermo). In total 1 μg of isolated gDNA was bisulfite converted, denatured, fragmented and hybridized to Infinium Methylation Bead Chip, following the manufacturer protocol (Infinium MethylationEPIC kit, Illumina). BeadChips were imaged using an Illumina Scan System and intensity was determined by iScan Control Software (Illumina). Sample intensities were normalized using functional normalization from the minfi package (v1.24.0)60 (link). Probes failing a detection p-value threshold (0.01) in at least 50% of samples were removed, as were probes identified as containing a SNP with a MAF >0.05. Differentially methylated probes were identified by applying limma (v3.34.3)54 (link) contrasts to M values (absolute change in beta value >0.1, FDR-corrected P-value < 0.05). Differentially methylated regions were identified using DMRcate (v1.14.0)61 (link) setting a threshold of absolute change in beta value in >0.1 and of Stouffer’s value in <0.05.
Other organizations :
University of Pittsburgh, Universidad de Navarra, Imperial College London, University of Miami, University of North Carolina at Chapel Hill, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Mayo Clinic, Universitat de Barcelona, UCLouvain, Lille Inflammation Research International Center, Hebron University, University College London, Consorci Institut D'Investigacions Biomediques August Pi I Sunyer, Texas A&M University, Medical University of Graz, Newcastle University
PureLink Genomic DNA Mini Kit (Thermo) for DNA extraction
Nanodrop (Thermo) for DNA quantification
Infinium MethylationEPIC kit (Illumina) for bisulfite conversion, denaturation, fragmentation, and hybridization
Illumina Scan System and iScan Control Software for imaging and intensity determination
Functional normalization from the minfi package (v1.24.0) for data normalization
Removal of probes failing detection p-value threshold (0.01) in at least 50% of samples and probes containing SNPs with MAF >0.05
Limma (v3.34.3) for identification of differentially methylated probes
DMRcate (v1.14.0) for identification of differentially methylated regions
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