Transcriptome profiling of the Pacific oyster Crassostrea gigas
The Pacific oyster Crassostrea gigas used in this study was originally collected from the coastal region at Yeongheungdo in Incheon (South Korea); since then, we have continuously cultured the species in a laboratory of West Sea Fisheries Research Institute (Incheon, South Korea) with filtered sea water (30 practical salinity unit (psu)) adjusted to 20°C on a 14 h: 10 h light/dark cycle with aeration. All the experiments were approved by the animal care and use committee of National Fisheries Research and Development Institute (NFRDI, Pusan, South Korea).  We sequenced transcriptome of pooled tissues of C. gigas that were exposed to different culture conditions (i.e. temperature, salinity, pH). The pooled tissues were frozen in liquid nitrogen and homogenized with a glass pestle. Total RNA was extracted with the RiboPure kit (Applied Biosystems, Foster City, CA, USA) based on manufacturer's protocol. DNA digestion was performed using DNase I (Sigma, St. Louis, MO, USA). Total RNA was quantified by absorption of light at A260 and quality checked by analyzing the ratios A230/260 and A260/280 using a NanoDrop® 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Synthesis of the paired-end library and sequencing with Illumina HiSeqTM 2500 (Illumina, San Diego, CA, USA) were performed at the National Instrumentation Center for Environmental Management (NICEM, Seoul National University, Seoul, South Korea). Briefly, adaptor-ligated fragments were separated by size on an agarose gel, and the desired range of cDNA fragments (200 ± 25 bp) were excised from the gel. Proper fragments were selected and purified and subsequently PCR amplified to create the final cDNA library template. Image deconvolution, base calling, and quality value calculations were performed using the Illumina GA pipeline (ver. 1.6) according to the manufacturer's instructions.