Gene expression in MeWo cells after addition of recombinant protein His-Apo-R15
To investigate the effects of the recombinant RGD-apoptin protein on human melanoma cells (MeWo cell line), we performed gene expression analysis using RNA sequencing. Gene expression profiling was performed at two time points: 24 hours and 48 hours after adding the recombinant protein to MeWo cells. Overall design: MeWo cells were seeded in 24-well plates and were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) with antibiotics in humidified atmosphere with 5% CO2 at 37C until they reached 70% of confluence. Next, DMEM was replaced with OptiMEM medium containing recombinant protein or renaturation buffer. Recombinant His-Apo-R15 protein was added to the cells at a final concentration of 10 uM. As a control, OptiMEM with 0,2 M L-Arginine HCl, 50 mM Tris-HCl, pH 7,6 buffer (recombinant protein renaturation buffer, final concentration of L-Arg was 24 mM) was added to the cells. After 24 or 48 hours, RNA was isolated from the cells. The experiment was performed in three biological replicates. RNA was extracted using ExtractRNA reagent (Evrogen, Russia), according to the manufacturer protocol. Remaining genomic DNA was removed by Turbo DNAse (Invitrogen, USA). Total RNA (400 ng) was used for library preparation. Enrichment of polyadenylated RNA was performed with KAPA mRNA Capture Kit (Roche, Switzerland) and libraries were prepared using the KAPA RNA Hyper Kit (Roche, Switzerland), according to the manufacturer protocol. Subsequently, RNA cleanup was performed with the RNA Clean XP kit (Beckman Coulter, Brea, USA). The library underwent a final cleanup using the KAPA HyperPure Beads (Roche, Switzerland) after which the libraries size distribution and quality were assessed using a high sensitivity DNA chip (Agilent Technologies, USA). Libraries were pooled equimolarly and diluted to a final concentration of 750 pM. Sequencing was carried out on a NextSeq 1000 platform (Illumina, USA) using the NextSeq 1000/2000 P2 Reagents kit (200 Cycles) v3, with 4% PhiX (Illumina, USA) added as an internal control.