title: GSM3464036: S_M mRNA; Schistosoma japonicum; RNA-Seq
library_construction_protocol: Worms were recovered by hepatic-portal perfusion with PBS solution at five weeks post infection. The worms were transferred into sterile RPMI 1640 and separated by sex under light microscopy, after which the worms were washed three times with sterile RPMI 1640. Schistosomes were homogenized in a glass homogenizer, and total RNA was isolated using TRIzol reagent according to the manufacturer's instructions (Invitrogen, USA). A total amount of 3 μg total RNA per sample was used as input material for the subsequent RNA sample preparations. rRNA was depleted using Epicentre® Ribo-zero rRNA Removal Kit, and sequencing libraries were generated and RNA sequencing was performed according to the manufacturer's protocol using the NEBNext ® Ultra™ Directional RNA Library Prep Kit for Illumina (Illumina®, NEB, USA). Briefly, first strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase. Second strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP were replaced by dUTP. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of the preferred 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA), and the library quality was assessed on the Agilent Bioanalyzer 2100 system. The libraries were quantified with a Qubit 2.0 Fluorometer (Life Technologies, Grand Island, NY) and sequencing was performed on an Illumina HiseqTM 2500/MiSeq platform (Illumina).