title: Transcriptomic analysis of HepG2 cells exposed to extracellular citrate
design: Transcriptomic analysis of HepG2 cells exposed to extracellular citrate
library_construction_protocol: Human hepatocellular carcinoma cell lines HepG2 were cultured in Dulbecco's Modification of Eagle's Medium (DMEM), supplemented with 10% fetal bovine serum, 100 U/mL penicillin/streptomycin and 2 mM L-glutamine, at 37˚C with 5% CO2. All experiments were performed using cells with less than 10 passages, in the logarithmic growth phase.  Sodium citrate was dissolved in distilled water to obtain stock solutions (1 M). HepG2 cells were seeded into 6-well plates at a density of 5×105 cells per well and treated for 24 h at 37 °C with 10 mM sodium citrate.  Total RNA was extracted using Zymo Research, according to the manufacturer's instructions. The RNA was transcribed to cDNA using oligo (dT) primers and the GeneAmp RNA PCR Core Kit (Applied Biosystems) and the cDNA was amplified via real-time PCR using Power- SYBR Green PCR Master Mix (Promega) on the 7500 Fast Real-Time PCR System (Applied Biosystems). RNA concentration and purity were evaluated using NanoDrop™ 2000/2000c (Thermo Fisher Scientific, Waltham, MA, USA), whereas sample integrity was analyzed using Tape Station 4200 (Agilent Technologies, Santa Clara, CA, USA) using an RNA Screen Tape Assay. Indexed libraries were prepared from 1 µg/ea purified RNA using the TruSeq Stranded mRNA (Illumina, San Diego, CA, USA) Library Prep Kit according to the manufacturer's instructions. After the enrichment of mRNA using 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 1.5 nM.
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-13852:CITRATE_1_p