title: P865_205
organism: Homo sapiens
description: Protocols: BEAS-2B cells (European Collection of Cell Cultures) were cultured in bronchial epithelial cell growth medium (BEGM, Lonza) supplemented with BEGM bullet-kit (Lonza: recombinant epidermal growth factor (EGF), hydrocortisone, insulin, bovine pituitary extract, GA-1000 (Gentamicin Sulfate and Amphotericin-B), retinoic acid, transferrin, triiodothyronine and epinephrine). Cells were cultured in flasks and plates pre-coated with: 0.01 mg/mL fibronectin, 0.03 mg/mL bovine collagen type I, 0.01 mg/mL bovine serum albumin and 0.2% penicillin-streptomycin in BEGM additive free medium for 1-2 h prior to the seeding. Cells were maintained in a humidified atmosphere at 37˚C, 5% CO2 and sub-cultured at 80% confluency. For the long-term exposure, BEAS-2B were seeded in 6 well-plates (5000 cells/cm2, 2 mL cell medium per well) and allowed to attach for approx. 2 h. Thereafter, cells were exposed to 1 µg/mL Ag10 (approx. 0.2 µg/cm2) in triplicates. Cells were split (trypsinization), counted, reseeded twice a week (5000 cells/ cm2, 2 mL cell medium per well) and re-exposed to the Ag nanoparticles. For RNA extraction, the cells were washed after exposure and total RNA was extracted using the RNeasy Mini Columns (Qiagen) according to the manufacturer's instructions, including the purification step with DNase I treatment. Stranded mRNA-seq (Illumina TruSeq RNA HT with polyA-selection