Transcriptome analysis of Perna perna under anthracene contamination

Adults Perna perna mussels (7-9cm) were harvested from the aquaculture farm of the Federal University of Santa Catarina (UFSC) in Brazil, acclimated for 6 days in a aquarium (40 L) with filtered seawater, aerated and with air temperature controlled in 22 ºC. Mussels were feed once per day with commercial reef food (Phyto-plus A and Phyto-plus B, MICROBE-LIFT). except in the last 2 days to avoid bioaccumulation from the food. Later, mussels were randomly distributed to 8 aquariums with filtered seawater (4 L), each one with 3 mussels. For exposure procedure, ANT was dissolved in dimethyl sulfoxide (DMSO) and added to 4 of 8 aquariums, getting a final concentration of 1 mg/L in 0.01% (v/v) of DMSO concentration. The other 4 aquariums was added only filtered seawater with a DMSO concentration of 0.01% (v/v). The mussels were exposed for 24h. Gills of mussels were dissected and immediately submitted to total RNA extraction protocol of RNeasy mini kit (QIAGEN), following manufacturer's instructions. The total RNA samples from a same aquarium were pooled, obtaining a final number of 8 total RNA samples. Quantitation and quality control were made by Bioanalyzer (Agilent) and Nanodrop (Thermo Fisher Scientific). The cDNA library were prepared with TruSeq Stranded mRNA Sample Prep LS Protocol and sequenced paired reads of 100 pb in one lane (multiplex) of Illumina HiSeq 2500 Flow Cell v4 with HiSeq SBS Kit v4.