De novo transcriptome sequencing of Rapana venosa from 6 different developmental stages using Hi-seq 2500
Larva Culture and Sample Preparation  The R. venosa stocks were collected from Laizhou bay in Shandong province, parent R. venosa culture, spawning and larva rearing refer to the study of Dazuo Yang (Yang et al., 2007). Planktonic larva were cultured in 3.5m×5.2m×1.5m cement pools at 24.4-25.4°C with the denisity of 15/ml. Isochrysis galbana, Chlorella vulgaris and Platymonas subcordiformis were mixed as diet for Planktonic larva. And larvae from six major developmental stage: one spiral whorl stage, two spiral whorls stage, three spiral whorls stage, four spiral whorls stage(competent larva), post larva (Harding, 2006; Pan et al., 2013) were collected under microscope, frozen in liquid nitrogen quickly till use. Each stage have three repeats for RNA extraction, every repeat contains approximately 500mg larvae.   RNA Purification, Library Preparation, Hi-Seq 2500 Sequencing  Samples from each stage was used for RNA extraction followed by the instructions of a Trizol Kit (Invitrogen, USA). A series of detection was carried out to insure the qualification of RNA, we use 1.1% agarose gels electrophoresis to monitor the contamination and degradation, use the NanoPhotometer®spectrometer (IMPLEN, CA, USA) to check purity. RNA concentration was measured by Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA). We followed manufacturer’s protocol of NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) to prepare sequencing library. Briefly, mRNA was purified from 3µg of total RNA (A mixture of RNA from six developmental stages with an equal ratio) using magnetic beads with poly-T-Oligo-attached. Under the elevated temperature, divalent cations were used to perform the fragmentation in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA synthesis was carried out by using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA was synthesized by using RNase H and DNA Polymerase I. Exonuclease/polymerase activities make remaining overhangs convert into blunt ends. Then, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization after adenylation of 3’ ends of DNA fragments. The AMPure XP system (Beckman Coulter, Beverly, USA) was used to purify library fragments and preferentially select cDNA fragments of 150~200bp length,. Then size-selected, adaptor-ligated cDNA was added with 3µl USER Enzyme (NEB, USA) followed by 15 min at 37°C then 5 min at 95 °C. Then added Universal PCR primers, High-Fidelity DNA polymerase, Index (X) Primer to carry out PCR procedure. PCR products were purified by using AMPure XP system while library quality was assessed (Agilent Bioanalyzer 2100 system). The sequencing of library preparations was performed on Illumina Hiseq 2500 platform while paired-end reads were obtained.
A mixture of RNA from six developmental stages with an equal ratio