Normoxia1
Total RNA from Daphnia was extracted using the mirVanaTM RNA isolation kit (Applied Biosystems) and treated with DNase (Ambion) to remove contaminating genomic DNA. RNA quality was assessed using the Agilent 2100 Bioanalyzer system and samples with a RNA Integrity Number (RIN) greater than 9 were used for mRNA library construction. Briefly, the cDNA libraries were prepared using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, USA) following the manufacturer’s protocol. Index codes were ligated to identify individual samples. mRNA was purified from the total RNA using poly-T oligo-attached magnetic beads (Illumina, San Diego, USA), and fragmented RNA was subjected to first and second strand cDNA syntheses using random oligonucleotides and SuperScript II, followed by DNA polymerase I and RNase H. After 3’ end adenylation, Illumina PE adapter oligonucleotides were ligated to cDNA. DNA fragments that ligated with adaptor molecules were amplified using the Illumina PCR Primer Cocktail in a 15-cycle PCR reaction. Products were purified and quantified using the AMPure XP and the Agilent Bioanalyzer 2100 systems, respectively. Before sequencing, the libraries were normalized and pooled together in a single lane on an Illumina MiSeq platform. Paired-end reads, each of 150-bp read-length, were sequenced.