title: RNA-Seq Reveals Infection-induced Gene Expression Changes in the Snail Intermediate Host of the Carcinogenic Liver Fluke, Opisthorchis viverrini-Uninfected Bithynia
design: Two distinct pools of soft tissues from uninfected (UB; n = 4) and O. viverrini-infected snails (IB; n = 4) were subjected to isolation of total RNA using the Trizol reagent (Invitrogen), followed by DNase I treatment (Promega) according to the manufacturer’s instructions. The amounts and integrity of total RNA were determined using a 2100 BioAnalyzer (Agilent Technologies). Polyadenylated (PolyA+) RNA was purified from 10 µg of total RNA from each pool of uninfected and O. viverrini-infected snails using Sera-Mag oligo(dT) beads, fragmented to a length of 100-500 nucleotides and reverse transcribed to cDNA using random hexamers. The size-fractionated cDNA was end-repaired and adaptor-ligated according to the manufacturer’s protocol (Illumina). Ligated products of ~300 bp were excised from agarose gels and PCR-amplified (15 cycles). Products were cleaned using a MinElute column (Qiagen, Netherlands) and paired-end sequenced on an Illumina HiSeqTM 2000 according to the manufacturer’s protocol.