Role of ABCC6 transporter in the dynamics of cell-cell and cell-matrix interaction
Role of ABCC6 transporter in the dynamics of cell-cell and cell-matrix interaction
The human hepatocellular carcinoma cell line (HepG2) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/L glucose, supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/mL penicillin and 100 µg/mL streptomycin. Cells were incubated in a humidified incubator at 37°C under an atmosphere containing 5% CO2.  The cells were subcultured at preconfluent densities using 0.25% trypsin-EDTA. The Abcc6 knockdown HepG2 cell line was constituted using a Lentivirus shRNA knockdown vector system. The cell transfection was carried out according to the instructions of the manufacturer Cyagen Biosciences (Santa Clara, CA, USA) from whom the lentiviruses were purchased. The mRNA was extracted from the cells using TRIzol reagent (TRIzol Reagent, Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. RNA was stored at −80 °C until further processing. RNA concentration and purity were evaluated using NanoDrop™ 2000/2000c (Thermo Fisher Scientific, Waltham, MA, USA), whereas sample integrity was analyzed by TapeStation 4200 (Agilent Technologies, Santa Clara, CA, USA) using RNA ScreenTape Assay 2.7Indexed libraries were prepared from 1 µg/ea purified RNA with TruSeq Stranded mRNA (Illumina) Library Prep Kit according to the manufacturer's instructions. After enrichment of mRNA by oligo dT magnetic beads and fragmentation, cDNA synthesis was performed, followed by adapter ligation and PCR amplification. For library quantifications, TapeStation 4200 (Agilent Technologies) was used. Indexed libraries were pooled in equimolar amounts with a final concentration of 2 nM.