De-novo transcriptome analysis of intestinal fluke Fasciolopsis buski
Transcriptome library for sequencing was constructed according to the Illumina TruSeq RNA library protocol as outlined in “TruSeq RNA Sample Preparation Guide”. The integrity of total RNA was verified using Agilent 2100 with an RNA integrity number. An Oligo Tex mRNA minikit (Qiagen) was used to isolate poly (A) mRNA after total RNA was collected from a single individual fluke (~100mg tissue) using TRI reagent® (Sigma) and following manufacturer’s protocol. Briefly, mRNA was purified from 1 μg of intact total RNA using oligodT beads (TruSeq RNA Sample Preparation Kit, Illumina). The purified mRNA was fragmented for 6 min at elevated temperature (94oC) in the presence of divalent cations and reverse transcribed with Superscript II Reverse transcriptase by priming with Random Hexamers. The Second strand cDNA was synthesized in the presence of DNA Polymerase I and RNaseH. The cDNA was cleaned up using Agencourt Ampure XP SPRI beads (Beckman Coulter). Illumina Adapters were ligated to cDNA molecules after end repair and addition of A base. Solid Phase Reversible Immobilization (SPRI) cleanup was performed after ligation. The library was amplified using 11 cycles of PCR for enrichment of adapter ligated fragments. The prepared library was quantified using Nanodrop (Agilent) and validated for quality by running an aliquot on High Sensitivity Bioanalyzer Chip (Agilent)