Transcriptome Analysis and Discovery of Genes Involved in Immune Pathways from Coelomocytes of sea cucumber (Apostichopus japonicus) after Vibrio splendidus Challenge
Coelomic fluid were extracted and centrifuged at 3000rpm for 2min to harvest the coelomocytes. Total RNA was extracted using TaKaRaMiniBEST Universal RNA Extract Kit (Takara, Dalian). Approximately 10 ug of total coelomocyte RNA was subjected to isolate Poly (A) mRNA with poly-T oligo attached magnetic beads (Invitrogen). Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNA-Seq sample preparation kit (Illumina, San Diego, USA), the average insert size for the paired-end libraries was 300 bp (AR50 bp). And then we performed the paired-end sequencing on an Illumina Hiseq2500 at the (LC Sceiences,USA) following the vendor's recommended protocol.