description: Protocols: HepG2 cells were cultured in DMEM, high glucose supplemented with 10 % fetal calf serum, 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) for gene expression analysis. 24 h after seeding, medium was changed to minimal essential medium (MEM) supplemented with 1 % charcoal-stripped FCS, penicillin (100 U/mL) and streptomycin (100 μg/mL). After further 24 h, medium was changed again to MEM supplemented as described above now additionally containing 0.1 % DMSO and the test compounds (10 µM garcinoic acid, 10 µM garcinoic acid + 10 µM GW9662, 1 µM pioglitazone, or 10 µM garcinoic acid + 1 µM pioglitazone) or 0.1 % DMSO alone as control. Each treatment condition was set up in four independent biological repeats (n=4). After 8 h incubation, cells were harvested (trypsin+EDTA), washed twice with cold phosphate buffered saline and then directly used for RNA extraction. RNA was extracted by the E.Z.N.A.® Total RNA Kit I (R6834-02, Omega Bio-Tek Inc., Norcross, GA, USA) according to the manufacturer's protocol. 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™ RNA Library Prep Kit for Illumina® (New England Biolabs (NEB), Ipswich, MA, U.S.A.) 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, adaptorligated 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.
attributes.tag: ENA first public
attributes.value: 2021-04-16
attributes.tag: ENA last update
attributes.value: 2021-04-06
attributes.tag: External Id
attributes.value: SAMEA8521192
attributes.tag: INSDC center alias
attributes.value: Goethe University Frankfurt
attributes.tag: INSDC center name
attributes.value: Goethe University Frankfurt
attributes.tag: INSDC first public
attributes.value: 2021-04-16T00:26:20Z
attributes.tag: INSDC last update
attributes.value: 2021-04-06T14:55:00Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10328:GARCINOIC_ACID_4
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HepG2
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: juvenile stage
attributes.tag: disease
attributes.value: hepatocellular carcinoma
attributes.tag: organism part
attributes.value: liver
attributes.tag: sample name
attributes.value: E-MTAB-10328:GARCINOIC_ACID_4