There were three replicates for each temperature treatment. Population identity was maintained at all times through the separation of populations into floating mesh tubs within the same tank so that northern barramundi (N) could be distinguished from southern barramundi (S) at cool (N22 from S22), control (N28 from S28) and hot (N36 from S36) temperatures. During growth trials fish were reared for a period of approximately 3.5 months (106 days), Akt inhibitor while at all times water chemistry, dissolved oxygen (> 5 mg/ml), pH, and temperature (experimental conditions ± 1 °C) were rigorously maintained. After this time, fish were humanely

sacrificed in accordance with Animal Ethics Permit A1249 and their weight was recorded as a measure of growth over the rearing experiment. White muscle tissue was chosen for gene expression analysis due to its growth responsiveness and high metabolic rate and was immediately dissected from each fish and snap frozen in liquid nitrogen. Total RNA was extracted by homogenizing frozen muscle tissue in Ultraspec solution (Biotecx; http://www.biotecx.com) using

a PRO200 homogenizer (PRO scientific Inc.; http://www.proscientific.com). RNA was precipitated in a solution containing 0.5 vol of RNA precipitation solution (1.2 M sodium chloride, 0.8 M disodium citrate) (Sambrook and Russel, 2001) and 0.5 vol of isopropyl alcohol. RNA quality and quantity were verified using a Nanodrop spectrophotometer (Nanodrop technology; http://www.nanodrop.com) via examination

MG-132 datasheet of absorbance ratios at OD 260/280 (range 1.98–2.06) and OD 260/230 (range 1.96–2.07) and by the visual inspection of the 18S Meloxicam and 28S ribosomal bands (and possible DNA contaminants) on a 1.5% agarose gel. After Nanodrop quantification, four RNA pools were constructed by combining individual fish RNA samples representing northern barramundi reared at 22 °C and 36 °C, and cool-adapted barramundi reared at 22 °C and 36 °C. Each sample pool consisted of 5 μg of total RNA from a total of eight separate individuals so that any potential variation between individual fish could be captured. Each RNA pool was then treated with a Turbo DNA-free kit (Ambion; http://www/invitrogen.com) as a precaution to eliminate trace DNA contamination before being sent for further processing including sample quality and quantity verification on an Agilent RNA Bioanalyzer chip directly prior to sequencing on an Illumina Genome Analyzer IIx (Macrogen Inc.; http://www.macrogen.com). Four mRNA-seq libraries were constructed representing pooled samples from northern and southern populations of barramundi reared at 22 °C and 36 °C incorporating unique bar-coding for each pool library. Illumina transcriptome mRNA pair-end sequencing (101 bp reads) was performed using standard protocols and reagents according to the manufacturer’s recommendations (Illumina Inc.; http://www.illumina.com).