RNA-seq to investigate the effect of SA112 a novel PPAR ligand in HepG2 cells
HepG2 cells (DSMZ) were cultured in DMEM, high glucose supplemented with 10% fetal calf serum (FCS), sodium pyruvate (1 mM), penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 °C and 5% CO2 and seeded in 6-well plates (1.0×106 cells/well). At 24 h after seeding, the medium was replaced with minimal essential medium (MEM) containing 1% charcoal-stripped FCS, penicillin (100 U/mL), and streptomycin (100 µg/mL). After 48 h, the medium was again exchanged for MEM with the same additives as before, additionally containing 0.1% DMSO and compound 2 (20 µM) or 0.1% DMSO alone as a control. Each treatment was performed in four biologically independent samples (n = 4). After 12 h incubation, total RNA was extracted. Cells were harvested, washed twice with cold phosphate buffered saline (PBS), and used for RNA extraction with the E.Z.N.A.® Total RNA Kit I (R6834-02, Omega-Bio-Tek Inc., Norcross, GA, USA). A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra TM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer's instructions.