Illumina HiSeq 2500 paired end sequencing; RNA-seq of cytoplasmic and nucleoplasmic samples of a time-course of Ad5-infected A549 cells
RNA-seq of cytoplasmic and nucleoplasmic samples of a time-course of Ad5-infected A549 cells
Human lung carcinoma A549 cells (ATCC CCL-185; American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle medium (Sigma, St. Louis, MO, USA) supplemented with 0.11 g/L sodium pyruvate, 10% fetal bovine serum (PAN Biotech, Aidenbach, Germany) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, PAN Biotech), and maintained at 37°C in a 5% CO2 atmosphere. Cells were routinely checked for mycoplasma contamination. For infections, the HAdV-C5 H5pg4100 virus was used, which has a deletion in the E3 region but a wild type phenotype in cell culture. Subconfluent A549 cells were infected at a multiplicity of infection of 50 fluorescence-forming units/cell for 6 to 48 h in all experiments. Mock-infected and HAdV-C5-infected cells (~2.5 × 10^6 per condition) were harvested by trypsinization (at 6, 12, 24 and 48 h p.i.), washed, pelleted and resuspended in 100 µl cold NP-40 lysis buffer (10 mM HEPES (pH 7.8), 10 mM KCl, 20% (v/v) glycerol, 0.25% (v/v) NP-40 and 1 mM DTT) and incubated on ice during 2 min. Samples were centrifuged (470 × g, 5 min at 4ºC) and the supernatants (~80 μl) were transferred into a new tube containing 600 μl TRIzol (Invitrogen, Carlsbad, CA, USA) and stored as the cytoplasmic fraction at -20°C. The remaining nuclear pellets were washed with cold NP-40 lysis buffer, centrifuged again and the supernatants were discarded. These residual pellets, which contain the cell nuclei were resuspended in 600 μl of TRIzol and stored as the nucleo-plasmic fraction at -20°C until further use. After collecting all samples, they were thawed and incubated for 5 min at room tem-perature, before 120 µl chloroform was added, tubes were shaken for 15 sec and incubated for 2-3 min at room temperature. Next, all samples were centrifuged (12,000 × g, 15 min at 4ºC) and the upper aqueous phase was transferred into a new tube. RNAs were pre-cipitated from these samples by adding 1.5 times the volume of 100% ethanol. From this mixture, 700 µl were transferred to RNA extraction columns (RNeasy Plus Mini Kit (Qiagen, Hilden, Germany)) and further processed according to the manufacturer's in-structions. Final RNA concentrations were measured with a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and samples were stored at -80°C. RNA samples from both cytoplasmic and nucleoplasmic fractions were sent to the Leibniz Institute of Virology NGS technology platform. Here, the RNA quality was evaluated using a Bioanalyzer with the RNA Nano Chip (Agilent Technologies, Böblingen, Germany). To isolate mRNAs from the total RNA sample, a poly(A) selection was performed using the NEBnext poly(A) mRNA Magnetic Isolation Module (NEB sequencing, Ipswitch, MA, USA). The cDNA libraries were generated with the ScriptSeq v2 RNAseq Kit (Epicentre Biotechnologies, Madison, WI, USA) using their strand-specific protocol where forward strands were labeled. The sizes and qualities of the libraries were controlled using the Bioanalyzer High Sensitivity DNA Chip Kit (Agilent Technologies).