title: GSM3030958: WGBS_PBL_E013; Homo sapiens; Bisulfite-Seq
library_construction_protocol: SA and VA samples - In brief, freshly isolated VAT and SAT samples were weighed and minced with surgical scissors. For each gram of minced tissue, 1.25 mg/mL of Collagenase I (Sigma Aldrich, CAT#C6885-1G) was added to 2 mL of HEPES buffer containing 7.9 g/L NaCl, 323 mg/L CaCl2.2H2O, 308 mg/L MgSO4.7H2O, 154.3 mg/L Na2HPO4 (anhydrous), 5 mL/L of 1M Hepes (Sigma Aldrich), 450 mg/L D-glucose, pH 7.2 – 7.4, 20 g/L BSA. The tissue was incubated for 30-45 minutes at 37ºC before being diluted 1:9 with additional HEPES buffer. The mixture was passed through a 250 micron mesh before being centrifuged at 300 rcf for 6 minutes at room temperature. The top lipid layer was removed and the adipocytes collected for snap-freezing in liquid nitrogen.; VATsamples - Snap frozen in liquid Nirtogen; PBL - 10 mL blood was collected into K2EDTA vacutainers. Following centrifugation at 1,300 rcf for 10 minutes at room temperature, the top plasma layer was removed and the buffy coat layer collected. Cells were washed three times, with vigorous resuspension, in 10 mL Tris-EDTA buffer (100 mM Tris-HCl, 0.1 mM EDTA), with pellets collected after centrifugation at 10,000 rcf for 10 minutes. The final pellet was resuspended in 500 µL of buffer and stored at -80ºC. DNA was extracted: SAVA - using proteinase K digestion and phenol chloroform extraction; VAT - DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Cat#69504); PBL - DNA was extracted from PBL sample types following the Gentra Puregene Blood Cell Kit (Qiagen, Cat#158445). Whole genome bisulfite sequencing libraries were prepared following Illumina's “Whole- Genome Bisulfite sequencing for Methylation Analysis” protocol.  Briefly, 1 mg of genomic DNA was spiked with 0.5% unmethylated lambda DNA and sonicated to generate fragments of size between 150 to 300bp. Library preparation was performed for core samples using the Illumina's Paired-end DNA Sample Prep Kit (discontinued, Illumina, CA, USA) according to the manufacturer's protocol. The size selected libraries were then subjected to bisulfite conversion as previously described {Clark, 2006 #385}. Adaptor-ligated bisulfite treated DNA was enriched by 10 cycles of PCR amplification using the PfuTurbo Cx Hotstart DNA Polymerase (Stratagene). Qualitative and quantitative checks of the libraries were performed using Agilent's High sensitivity DNA kit (Agilent) and KAPA Library quantification kit (KAPA Biosystems).