GSM7315634: Skin tissue, Atopic Dermatitis, NonLesion, rep 18; Homo sapiens; RNA-Seq
Biopsy sample collection: Lesional and nonlesional (>10cm from active lesions) truncal skin biopsies were obtained from AD patients, while healthy skin samples were taken from healthy controls using either a punch biopsy or a simple fusiform biopsy under Institutional Review Board-approved protocols. The samples were collected and stored in RNA protect Tissue Reagent (Qiagen Mat. No.1017980) before being frozen at -80°C. The biopsy sites showed no signs of infection. Blood protein quantification: 18 ml of serum were collected individually from each patient and healthy control using two Vacuette tubes (#455051) containing 9 ml of NH Sodium Heparin. The collected samples were centrifuged at 12000 rpm for 10 minutes at room temperature with a slow deceleration. The top layer of plasma was collected and then diluted with HBSS (Corning #21-020-CV) at a ratio of 1:1. The diluted blood was added above the Ficoll layer (4 ml, Cytiva #1714403), and centrifuged at 3000 rpm for 20 minutes at room temperature with a slow deceleration, which separated the PBMC. The PBMC was collected, diluted 2-fold with RPMI (Cytiva #SH30027.02), and then underwent another centrifugation at 2000 rpm for 10 minutes at room temperature. After resuspending the cells, 10% DMSO (Cell Signaling #12611S) was added to the sample, which was then frozen. To extract the RNA, the samples were thawed in a water bath, centrifuged to remove the supernatant, and then processed using the RiboPure Kit (Invitrogen). Finally, 1 μl of the sample was analyzed using the Agilent 2100 Bioanalyzer and RNA 6000 nano assay. 1000 ng of each sample was used to construct a library with Universal Plus mRNA 243 Seq and NuQuant (TECAN). The library was then tested for quality and concentration using two systems: Qubit® 2.0 Fluorometer with its dsDNA High Sensitivity Kit and Agilent 2100 Bioanalyzer with its DNA 1000 assay.