title: Crassostrea gasar: diesel fuel water-accommodated fraction group
design: Total RNA from the gill and digestive gland of each oyster was individually isolated using TRIzol reagent (Invitrogen, UK) and purified with the Nucleospin RNA II Total RNA Isolation Kit (AbGene, UK) following the supplier's protocol with minor modifications. Briefly, 50100 mg of each tissue was mechanically disrupted in the presence of 1 mL TRIzol using a homogenizer (Tissue-Tearor, BioSpec Products). The TRIzol protocol was strictly followed until separation of the phases, with 200 L of the upper aqueous phase transferred to a new tube for on-column purification using the Nucleospin RNA II Total RNA Isolation Kit (AbGene, UK). RNA was finally eluted in 60 L of RNase-free water. Residual genomic DNA contamination was removed during the RNA Nucleospin cleanup using the DNase I digestion following manufacturers instructions (AbGene, UK). The integrity of the purified total RNA was assessed using formaldehyde agarose gel electrophoresis, and RNA quantity was determined using a NanoDrop ND-1000 spectrophotometer (ThermoScientific, UK). Equal quantities of purified total RNA (3 g) were pooled into 10 samples: gill and digestive gland for each of diesel WAF 24 h, diesel WAF 72 h, PHE, sewage and control groups. The pools were used for synthesis of non-normalized full-length double-stranded cDNA. cDNA libraries were constructed for each sample using the SMARTer PCR cDNA Synthesis Kit (Clontech, Paris) according to the manufacturer's instructions. Full-length cDNA templates were then amplified by long-distance PCR using the Advantage 2 PCR Kit (Clontech, Paris). To ensure that the PCR products were not over amplified, the optimal number of PCR cycles was determined according to the manufacturer's guidelines, which was verified by agarose gel electrophoresis. The products were purified with the DNA Clean & Concentrator5 Kit (Zymo Research, USA). The amplified cDNA libraries were verified for quality by microcapillary electrophoresis (Agilent Bioanalyzer 2100, Agilent Technologies) and quantified using a NanoDrop ND-1000 spectrophotometer (ThermoScientific, UK). The 10 cDNA libraries were then combined into 4 samples. Both gill and digestive gland tissue-specific libraries were combined for each of the controls, sewage, PHE and diesel WAF (with both 24 and 72 h samplings). The libraries were then submitted to size-selection in a gel with two libraries prepared: one selected for larger fragments (cut from just below the 4 kb and sheared by nebulisation down to 500700 bp) and one for smaller fragments (cDNA fragments of approximately 700 bp). Molecular Identifier (MID) tags were used to enable subsequent identification of size-selected portions from each library dataset. The 8 tagged libraries (two for each cDNA library) were then combined and pyrosequenced on the half of a PicoTiter Plate using the 454 Genome Sequencer FLX System according to the manufacturers' instructions (454/Roche, http://www.454.com). Pyrosequencing was performed by the Centre for Genomic Research at the University of Liverpool.