Transcriptome of the adult stage
A normalized, complementary DNA (cDNA) library was constructed (EurofinsMWGOperon, Ebersberg, Germany; www.eurofinsdna.com). In brief, total RNA was extracted from three adult worms using the mirVana microRNA isolation kit (Ambion), and polyadenylated (polyA+) RNA was purified from 25 μg of pooled total RNA. First-strand cDNA synthesis from polyA+ RNA was primed using a hybrid, random hexamer (N6) oligonucleotide and transcribed using M-MLV H− reverse transcriptase. After RNA hydrolysis, a specifically designed adapter primer was attached to the 3′ end of the first-strand cDNA (5′-AGTCAGGACCTTGGCTGTCACTC-3′). The adapter sequences on both ends of the cDNA were then used to synthesize second-strand cDNA and to amplify (16 cycles) the cDNA employing oligonucleotides complementary to the adapters by long and accurate PCR (LA-PCR). Subsequently, specific adapter sequences A (5′-AGTCAGGACCTTGGCTGTCACTC-3′) and B (5′-ACCGGACAGTGAGTGACGCT-3′) (FLX Titanium, Roche) were added to the 5′ and 3′ ends of the cDNA, respectively. Normalization was conducted using one cycle of denaturation and reassociation of the cDNA. Reassociated double-stranded cDNA was separated from the remaining singlestranded cDNA (ss-cDNA, normalized cDNA) by purification on a hydroxylapatite column. The ss-cDNA was amplified (9cycles) using LA-PCR. The cDNA was size-selected (500–700 bp) following agarose gel electrophoresis and excision from the gel.