title: GSM6012638: VSMCs sample 9; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was isolated from cultured HAVSMCs with the commercially available NucleoSpin® RNA kit (Macherey-Nagel REF 740955) with several modifications. Briefly, media was removed and 300 μL QIAzol Lysis Reagent (QIAGEN Cat No. 79306) was added to each well of a 6-well plate. A cell scraper was used to scrape cells in QIAzol and cells from each well were collected into individual 1.5 mL tubes. Cells were vortexed and left to sit at room temperature for 5 minutes. To each tube, 60 μL of chloroform was added and cells were vortexed and left to sit for 2 – 3 minutes at room temperature. Cells were then centrifuged at 12,000 X g for 15 minutes at 4°C. The aqueous phase was removed to new tubes and 150 μL isopropyl alcohol was added to each. Following a 10-minute incubation at room temperature, each lysate was loaded onto a NucleoSpin® RNA Column and NucleoSpin® RNA protocol 5.1 was completed beginning at step five. Following elution, RNA was quantified with a NanoDrop™ 2000 Spectrophotometer (Thermo Scientific) and samples were stored at -80°C. RNA libraries were prepared for sequencing using standard Illumina protocols