Transcriptome Profiling of Hypoxia Adaptation

Transcriptome profiling using Next Generation Sequencing (NGS) was performed for individuals exposed to normoxia and hypoxia for one week. Three replicate pools of males (n = 10) for each treatment were fast frozen in liquid nitrogen, and immediately stored at −80 °C. Total RNA was isolated using a PureLink RNA Mini Kit (ThermoFisher Scientific, UK). TruSeq RNA libraries (Illumina, USA) were synthesised and sequenced using 100 base paired-end sequencing (HiSeq 2000, Illumina, USA). Details of assembly, annotation and data availability are provided in^{55 (link)}. Alignment of the assembled transcriptome to the original reads was performed using Bowtie v.1.1.1^{56 (link)}. Gene counts were generated by RSEM v.1.2.29 and imported into R v.3.3.1 using Tximport v.1.0.3. Differential gene expression analysis was performed at the level of Trinity “genes” using DESeq2.v.1.12.4^{57 (link)} to identify haemocyanin genes that displayed significantly different expression (P < 0.05) between the hypoxic and normoxic treatment.