Illumina HiSeq 2500 paired end sequencing; RNA-Seq of BEAS-2B cells treated with low doses of Ni and NiO nanoparticles as well as NiCl2 for 6 weeks
RNA-Seq of BEAS-2B cells treated with low doses of Ni and NiO nanoparticles as well as NiCl2 for 6 weeks
After treatment, cell samples were collected by trypsinisation.  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 0.5 µg/mL Ni and NiO nanoparticles or NiCl2 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 Ni or NiO nanoparticles or NiCl2.  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)
Experimental Factor: compound
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