title: RNA-seq of Euryale ferox
design: To construct the cDNA libraries, we used 3 ŚĚg total RNA per sample as the input material. The NEBNext? Ultra? RNA Library Prep Kit for Illumina? (NEB, USA) was used to generate a series of corresponding libraries, and index codes were applied to every sample. Briefly, mRNA was purified from total RNA using oligo dT magnetic beads. RNA fragmentation was carried out at high temperature in the presence of divalent cations in NEBNext First Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized using random hexamer primers and M-MLV Reverse Transcriptase (RNase H-), and the second strand was synthesized using DNA Polymerase I and RNase H. The exonuclease/polymerase activities were used to convert the remaining single-stranded overhangs to blunt ends. After adenylation of the 3ĄŻ ends of the DNA fragments, the NEBNext Adaptor containing a hairpin loop structure was added. cDNA fragments between 150 and 200 bp were purified using the AMPure XP system (Beckman Coulter, Beverly, USA). Afterwards, the 3 ŚĚl USER Enzyme (NEB, USA) was made use of picking size, adaptor-ligated cDNA at 37ĄăC for 15 min followed by 5 min at 95ĄăC before PCR. The PCR were performed using universal PCR primers, Index (X) Primer and Phusion High-Fidelity DNA polymerase. The Agilent Bioanalyzer 2100 system was used to purify the PCR products and to evaluate the quality of the cDNA libraries. Paired-end DNA sequencing data for the 12 libraries was then generated on an Illumina Hiseq 2000 instrument.