cell line: SGBS preadipocyte cell line time: day 11 experiment: 2 replicate: 9 conversion: BS
Growth protocol
Human Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocytes were grown to confluency in Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12Nutrient Mixture (Ham) supplemented with 10 % fetal bovine serum (FBS), 33 µM biotin, 17 µM pantothenate, 100 U/ml penicillin, 100 µg/ml streptomycin, 1ng/µl fibroblast growth factor (FGF) 1, and 90 µg/ml heparin. Three days post-confluency (day 0), SGBS cells were washed three times with PBS and stimulated to differentiate with serum-free growth medium, supplemented with 10 nM insulin, 200 pM triiodothyronine, 1 µM cortisol, 2 µM rosiglitazone/BRL49653, 500 µM 1-methyl-3-isobuthylxanthine (IBMX), 250 nM dexamethasone (Dex), and 0.01 mg/ml human transferrin. After 6 days, FGF-1, Heparin, IBMX, Dex, rosiglitazone were removed from the medium. Cells were harvested at day 0, 1, 7 and 11.
Extracted molecule
genomic DNA
Extraction protocol
DNA was extracted using the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions, including an RNase A digest (RNase A solution, Qiagen) and Proteinase K digest (Puregene Proteinase K, Qiagen). DNA quality was checked by Agilent Bioanalyzer ( Agilent Technologies, Santa Clara, CA, USA) and quantified by Qubit fluorimetry (Thermo Scientific, Wilmington, USA).
Label
Cy5 and Cy3
Label protocol
Standard Illumina Protocol
Hybridization protocol
DNA samples (0.7-1.5 ng) were bisulfite (BS)- and oxidative bisulfite (oxBS)-treated using the TrueMethyl Array Kit (CEGX) according to the manufacturer’s instructions in the User Guide Version 2 (Feb 2015) as described by Gross et al. (2017) and hybridized to Illumina Infinium Human Methylation450 Beadchip using standard Illumina protocol.
Scan protocol
Arrays were imaged using BeadArray Reader using standard recommended Illumina scanner setting.
Description
Day11_rep09_BS
Data processing
Quality control, data preprocessing and data normalization were done using the RnBeads R package. Briefly, raw IDAT files were used as input and preprocessed separately for BS and oxBS data. Probes were filtered for a detection p-value threshold of 0.01, cross-reactive probes were removed (Chen et al., 2013) and remaining data was normalized using the beta-mixture quantile normalization (BMIQ) method. Data represent BS and oxBS beta values after filtering for low quality and cross-reactive probes and performing beta-mixture quantile normalization (BMIQ).
Metabolic changes during human adipocyte differentiation promote stable DNA hydroxymethylation and binding of acetylated NEIL1 to adipogenic enhancers [450k array]
