Smoking effect on DNA methylation in human blood CD8T cells

To better characterize smoking–associated methylation changes in human blood CD8T cells, we used Illumina HumanMethylation450 and HumanMethylationEPIC BeadChips to assess DNA samples from smokers (SM, n=61) and never smokers (NS, n=71). As part of the Epigenetic Biomarkers of Tobacco Smoke Exposure project, a group of healthy volunteers (61 smokers and 71 nonsmokers) was recruited at the NIEHS Clinical Research Unit (protocol 10-E-0063) between March 2013 and January 2018 from the Raleigh, Durham and Chapel Hill, NC area. All nonsmokers were self-reported as not having smoked >100 cigarettes in their lifetime. Smokers reported their average daily cigarette consumption for the past 3 months [Su D et al. 2016]. For each individual, CD8 T cells were isolated from whole blood using anti-CD8+ antibody-coated magnetic beads. Nucleated DNA from CD8T cells was collected after sucrose-induced osmotic lysis of cells followed by phenol-chloroform extraction and DNA was stored in Tris-EDTA buffer at -20°C. The HumanMethylation450 and HumanMethylationEPIC BeadChips (Illumina) were used to measure methylation by the NCI Center for Genome Research. Specifically, 500 ng of DNA was bisulfite converted using the EZ-DNA Methylation kit (Zymo Research), hybridized to HumanMethylation450 BeadChip arrays and then scanned with an iScan microarray scanner (Illumina) following the manufacturer’s protocols. The raw IDAT files of 450K and EPIC methylation arrays were read into R with the minfi package [Aryee MJ et al. 2014] separately; the combineArrays function in minfi was utilized to combine the two arrays’ data together based on their common CpG sites. Then, the data was preprocessed with background and dye bias correction using the preprocessNoob method [Fortin JP et al. 2017] and Methylation β value for each CpG site in each sample was output for further analyses.