Illumina HiSeq 2500 sequencing; RNAseq of human epithelial lung carcinoma cell line A549 exposed to ceria, silver or zinc ions, micron-sized particles or nanoparticles
RNAseq of human epithelial lung carcinoma cell line A549 exposed to ceria, silver or zinc ions, micron-sized particles or nanoparticles
A549 cells were purchased from ATCC (VA, USA). A549 is an epithelial lung adenocarcinoma cell line established in 1972 (Giard et al., 1973).  The cells were cultured in tissue culture flasks (75 cm2 and 275 cm2) in RPMI 1640 medium with Glutamax (Gibco, ThermoFisher Scientific Inc., Landsmeer, the Netherlands) supplemented with 10% Fetal Bovine Serum (FBS, Greiner BioOne Serum, Greiner BioOne BV, Alphen aan de Rijn, the Netherlands) and 1% penicillin/streptomycin (Gibco, 100 IU penicillin per mL, 100 µg streptomycin per mL). Cells were culture at 37oC in a humidified atmosphere of 5% CO2 in air. The adherent cells were harvested by a short incubation with 0.5% EDTA trypsin in Ca/Mg free Dulbecco's Phosphate Buffered Saline (Gibco). 8x10 to the 5 cells were seeded and cultured in wells of a 6 well plate for 18 hours, after which the cells were exposed ZnCl2 6h : A549 cells were exposed to ZnCl2 at 24.6 micrograms per ml for 6 hours. After this, the cells (approximately 2 million per well) were quickly washed with PBS (phosphate buffered saline) twice at room temperature after which the 6 well plates were deep frozen by quenching on liquid nitrogen (-196oC) and stored at -80oC until extraction for omics evaluation Total RNA was extracted from A549 cells using a micro RNeasy Kit (Qiagen, Crawley, UK) according to the manufacturer's protocol. RNA was quantified with a NanoDrop 8000 spectrophotometer (Thermo Scientific, Waltham, MA), and the integrity of RNA was evaluated with a Agilent 2200 Tapestation (Agilent Technologies, Santa Clara, CA). Only RNA with integrity numbers (RINs) greater than 7.0 were used for subsequent RNA-seq experiments. All RNA libraries were produced using the Biomek FxP (Beckman Coulter A31842) with Ultra Directional RNA Library Prep Kit (New England Biolab E7420L) and NEBnext Multiplex Oligos for Illumina Dual Index Primers (New England Biolabs E7600S), using provided protocols and 500ng of total RNA. Constructed libraries were assessed for quality using the Tapestation 2200 (Agilent G2964AA) with High Sensitivity D1000 DNA screentape (5067-5584), and quantified using Kapa Library Quantification Kit (Kapa Biosystems KK4824) on an AriaMx Realtime PCR System (Agilent G8830A).