Fresh Water eel MRC 2
Total RNA was extracted from the cells using the mirVanaTM isolation kit (Applied Biosystems) and treated with DNase (Ambion) to remove contaminating genomic DNA. Agilent 2100 Bioanalyzer system was used to assess RNA quality of samples and the samples with a RNA Integrity Number (RIN) greater than 9 were used for transcriptome library construction. cDNA library wasconstructed using TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina), each prepared from 300ng of total RNA. The cDNA libraries were constructed according to manufacturer’s instruction and index codes were ligated as identification to individual samples as previous described. Briefly, mRNA was purified from the total RNA using poly-T oligo- attached magnetic beads (Illumina, San Diego, USA) to remove the ribosomal RNA. Then the Mrna was fragmented by using divalent cations in Illumina proprietary fragmentation buffer at 94 ?C for 1 min. First strand cDNAs were synthesized using random oligonucleotides and SuperScript II, then and second cDNAs were synthesized using DNA polymerase I and RNase H. Overhangs were blunted by using exonuclease/polymerase, followed by 3’ end adenylation. After adenylation of 3’ ends of DNA fragments, Illumina PE adapter oligonucleotides were ligated to DNA fragments. DNA fragments that ligated with adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycles PCR reaction. Libraries were purified using AMPure XP system and quantified using the KAPA Library Quantification Kits. Before submitted to sequencing, the libraries were normalized and pooled together in a single lane on an Illumina MiSeq platform and 150 bp paired-end reads were generated.