title: Octopus vulgaris hemocytes transcriptome. Healthy octopus without coccidiosis by Aggregata octopiana
design: Specimens of O. vulgaris naturally infected by A. octopiana were collected by traps, an artisanal fishing gear used by local fishermen from the Ria of Vigo, Spain (24° 14.09’N, 8° 47.18’W). Octopuses were maintained in tanks filled with filtered sea water at 15 °C during 24 h. After anesthesia, a dorsal incision was made through the skin and mantle muscle behind the head to withdraw hemolymph with a disposable syringe (1 ml) inserted directly into the cephalic aorta. One milliliter of hemolymph from each octopus was centrifuged at 12000 × g, 4 °C for 5 min. The pellet of hemocytes was re-suspended in 1 ml of Trizol reagent (Invitrogen) and stored at -80 °C until the analysis was performed. The digestive tract from each octopus was dissected and analyzed for the Aggregata octopiana infection. Taking into account both parameters, the intensity of infection and the histopathological damage, octopus were divided in two groups: the first one, showing a high parasite load, high intensity of infection and strong histological caecum damage; termed sick octopus group; and the second one, having a null or low parasite load, low intensity of infection and without histological caecum damage; termed healthy octopus group. Total RNA from the hemocytes of 5 sick and 5 healthy octopus selected from each group was extracted according to the Invitrogen protocol. A total of 1.5 μg of RNA from each of the 5 animals per group was pooled to construct the mRNA libraries according to the Illumina standard protocol. Thus, two mRNA libraries (one from the pool of sick octopus, and one from the pool of healthy octopus) were analyzed in a Genome Analyzer (GAII). In short, mRNA was purified using oligo (dT) probes and then fragmented into small pieces using divalent cations under a high temperature. The cleaved RNA fragments were used for first strand cDNA synthesis using random primers, modified and enriched for attachment to the Illumina flow cell. The two hemocyte libraries were generated using the mRNA sequencing sample preparation kit (Illumina). The libraries were validated by processing an Agilent DNA 1000 chip on a 2100 Bioanalyzer (Agilent) and quantified by qPCR using complementary primers of the library adapters with the KAPA SyBR FAST Universal qPCR kit (KAPA Biosystems). The cDNA libraries were sequenced on the Illumina sequencing platform (GAII equipped with a paired-end module) performing 105 cycles per read on two flow cell lanes.