title: GSM3018856: RR_RNA_VAT_E018; Homo sapiens; RNA-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). RNA-seq libraries were prepared using Illumina TruSeq Stranded Total RNA Library Prep Kit 500 ng of RNA was used for each sample and ribosomal RNA reduction was done using
attributes.tag: GEO Accession
attributes.value: GSM3018856