title: Illumina HiSeq 2000 sequencing; Tempo-Seq targeted RNA sequencing of the HepaRG cell line treated with aflatoxin B1, benzo[a]pyrene, cyclosporin A, rotenone and thrichostatin A over a range of chemical concentrations and exposure time points
design: Tempo-Seq targeted RNA sequencing of the HepaRG cell line treated with aflatoxin B1, benzo[a]pyrene, cyclosporin A, rotenone and thrichostatin A over a range of chemical concentrations and exposure time points
library_name: Sample 253_s
library_construction_protocol: After chemical exposure, hepatocytes of the HepaRG cell line were washed with PBS and lysed with TempO-Seq lysis buffer (TempO-Seq lysis buffer 2x diluted to 1x with PBS, BioSpyder Technologies, proprietary kit). Following cell lysis, the samples were frozen at -80° and shipped for analysis on dry ice. HepaRG were provided by Biopredic International (Rennes, France) in cryopreserved vials. Cells were maintained into 150 cm2 flasks for two weeks in culture medium consisting of William's MediumE (Thermo Fisher Scientific, Melegnano, Italy) with 10% FBS (HyClone Fetal- Clone III, HyClone), 1% l-glutamine, 1% penicillin/streptomycin, 5 μg/ml bovine insulin and 50 μM hydrocortisone (all from Sigma, Milan, Italy). The cells were then cultured in differentiation medium, which consists of maintenance medium plus 1.7% DMSO (starting with 0.85% DMSO for 1 day) for 2 weeks. Subsequently, the hepatocytes were selectively trypsinised and detached, whereas the biliary cells remained adhered to the plate. The hepatocytes were finally transferred into clear bottom black polystyrene 96-well plates (5 × 104 cells/well in 100 μL) to undergo chemical treatment.  The cells were treated in 9 replicates (3 biological and 3 technical replicates) with 8 concentrations of each chemical individually, diluted in cell media with the final concentration of DMSO corresponding to 0.1%. The chemical concentrations were:   • Aflatoxin B1 (Cas No. 1162-65-8, purity ≥ 99.2%, Sigma), benzo[a]pyrene (Cas No. 50-32-8, purity ≥ 99.0%, Sigma), cyclosporine A (CAS No. 59865-13-3, purity > 99.9%, Sigma), trichostatin A (Cas No. 58880-19-6, purity > 98%, Sigma): 0.08192, 0.2048, 0.512, 1.28, 3.2, 8, 20 and 50 µM  • Rotenone (CAS No. 83-79-4, purity > 95.9%, Sigma): 0.016384, 0.04096, 0.1024, 0.256, 0.64, 1.6, 4 and 10 µM  Following the chemical treatment, the samples were incubated for 2, 6, 12, 24 or 48 h. The study included vehicle controls and quality controls (MAQC).  All the procedures for cell seeding, serial dilution of chemical and cell treatment were fully automated using the robotic liquid handlers Hamilton Star and Starlet platforms (Hamilton Italia Srl, Agrate Brianza, Italy).  No nucleic acid extraction was performed Biospyder probe manifest Homo sapiens whole genome ver 1.2 was used (file: 180905Human_Whole_Transcriptome_1.2Manifest.txt). A standard attenuation was applied (file 190220 Human Whole Transcriptome 1.2 Attenuation.txt)