Illumina HiSeq 2000 paired end sequencing; ribo-depleted RNA-Seq of HepG2 cells exposed to Benzo[a]pyrene for 6,12,18,24,36 and 48h.
ribo-depleted RNA-Seq of HepG2 cells exposed to Benzo[a]pyrene for 6,12,18,24,36 and 48h.
Human hepatocellular carcinoma HepG2 cells obtained from ATCC (HB-6065) were used as a cell model.  HepG2 cells were cultured as a monolayer in 95% humidity, with 5% CO2 atmosphere at 37 ˚C.  The cells were cultured in a Minimal Essential Medium (MEM) with 10% of Fetal Bovine Serum (FBS), 1% penicillin/streptomycin, 1% sodium-pyruvate and 1% non-essential amino acids.  All reagents were obtained from Gibco BRL (Breda, the Netherlands).  Passages were done at pre-confluent densities by standard trypsin-EDTA solution.  Three hundred thousand cells were used in each well of a 6-wells microtiter plate.  At 80% confluence, the medium was replaced with fresh medium containing either 3 mM of BaP or by the control vehicule (0.5 % DMSO) during six different exposure time (6h, 12h, 18h, 24h, 36h and 48h) in two independent experiments (biological replicates). First, Total RNA was treated with the Epicentre Ribo-Zero kit (cat.#MRZH11124) to remove the all the rRNAs.  The remaining RNAs were processed using the TruSeq RNA Sample PrepKit according to the Illumina protocol.  After cDNA synthesis, 6.5 pmol of DNA from each sample were clustered and sequenced on the Illumina HiSeq 2000 (Illumina) in 100 bp paired-end reads.
Experimental Factor: time
48 hour
Experimental Factor: compound
none