gender: male subject: 1 age: 43 yr age group: young tissue: cultured peripheral blood mononuclear cells treatment: 50 uM WY14,643
Treatment protocol
Peripheral blood mononuclear cells were incubated in RPMI1640 medium with 2 mmol/L L-glutamine, 10% fetal bovine serum and antibiotics (penicillin and streptomycin) in the presence of 5% CO2 at 37°C at 1.0 × 10^6 cells per ml with either WY14,643 (50 μM) or vehicle (DMSO, 0.05%). After 13 hrs of exposure the cell suspensions were transferred to 15ml tubes and centrifuged for 5 minutes at 1,600rpm at 4°C. The two cell pellets per donor were re-suspended in ice-cold PBS, and each transferred to separate eppendorf tubes for RNA and DNA isolation and again centrifuged for 5 minutes at 5,000rpm at 4°C. After removing the supernatant, pellets for DNA isolation were snap frozen on dry-ice and stored at -80°C. The pellets for RNA isolation were resuspended in 700 μL of Buffer RPE with added B-mercaptoethanol according to manufacturer’s instructions (Qiagen) and passed five times through a 23G needle before freezing at -80°C.
Growth protocol
Peripheral blood mononuclear cells from ten healthy Caucasian male blood donors, aged 30, 31, 34, 35, 43, 52, 62, 64, 65 and 66 years, were isolated directly after arrival of the buffy coat (max. 8 hours after donation) by Ficol-paque Plus density gradient centrifugation (Amersham Biosciences, Roosendaal, the Netherlands). All donors gave full written informed consent.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted from the PBMC pellets by using DNeasy Blood and Tissue Kit (Qiagen), including RNAse treatment, according to the manufacturer’s instructions. All samples were quality tested on a Nanodrop spectrophotometer prior to proceeding with the microarrays.
Label
Cy3 and Cy5
Label protocol
DNAs were prepared in a total volume of 20 μl (1 μg of FF) using a commercial kit EZ DNA Methylation kit (catalog number D5001; Zymo Research Corp, Orange, CA).
Hybridization protocol
Bisulfite converted DNA was amplified, fragmented and hybridized to Illumina Infinium HumanMethylation450 Beadchip using standard Infinium HD Methylation Assay protocol
Scan protocol
Arrays were imaged using HiScan Reader using standard recommended Illumina scanner settings
Data processing
SWAN normalization on IDAT files using library minfi (v1.6.0) in R/Bioconductor, with beta-threshold = 0.001
