RNA-Seq cercariae allopatric host generation 3
RNAseq library construction was done with the TruSeq RNA Sample Seq kit (Illumina Inc., USA) was used according to the manufacturer's protocol. Briefly, poly-A containing mRNA molecules were purified from 70 to 450ng of total RNA using oligo-dT magnetic beads. The purified mRNA was fragmented by addition of the fragmentation buffer and was heated at 94°C in a thermocycler for 8 min. First strand cDNA was synthesised using random primers. Second strand cDNA synthesis, end repair, A-tailing, and adapter ligation was done in accordance with the manufacturer's instructions. Purified cDNA templates were enriched by 15 cycles of PCR for 10 s at 98°C, 30 s at 65°C, and 30 s at 72°C using Illumina's proprietary primers and Phusion DNA polymerase. Each indexed cDNA library was verified using a DNA 1000 Chip on a Bioanalyzer 2100 and quantified by real time PCR with the KAPA Library Quantification Kit for Illumina Sequencing Platforms (Kapa Biosystems Ltd, SA) then diluted to 10 nM using Illumina's resuspension buffer. Sequences were adaptor trimmed, cleaned and groomed.