RNA-seq experiment of V79 cells treated with UV light and incubated either in monoculture or coculture with untreated cells
RNA-seq experiment of V79 cells treated with UV light and incubated either in monoculture or coculture with untreated cells
The cells were sorted based on GFP fluorescence directly into RLT buffer to lyse them and prevent RNA degradation.  V79 chinese hamster pulmonary fibroblast cells were cultured in DMEM supplemented with 10 % fetal bovine serum (FBS) in humidified atmosphere with 5 % CO2 at 37 Â°C. Cells stained with cytoplasmic dye in green were treated with a combination of UV-C and UV-C light of 0.15 J/cm2 and seede in monoculture with eqally treated cells or in coculture with untreated cells. After 12 hours incubation, cells were sorted based on the green fluorescence.  Total cellular RNA was isolated using Qiagen RNEasy Mini kit, and DNA was removed by the addition of DNase I (New England Biolabs). Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.