The successful application of RNA seq technology in the vaccine zebrafish interaction model in this work established a new experimental plat form for investigating the vaccine specific host immune Olaparib IC50 responses in a comprehensive and sensitive manner. Fu ture studies using this approach will likely provide fur ther significant insights into the detailed mechanisms of teleost immunity that will benefit the aquaculture industry, both from economic and human food source perspectives. Methods Fish and immunization Healthy zebrafish, weighing 0. 3 0. 1 g and about 6 months of age, were obtained from the Inhibitors,Modulators,Libraries animal center at the East China University of Science and Technology and maintained at 22 2 C in a zebrafish cultivation system with a photo period of 12 12 h.

Aquaria were supplied with flow through dechlorinated and continuously aerated water at a rate of approximately 2��10 4 min 1. After at least one week of acclimatization, they were randomly divided into six treatment groups in cluding three immunized groups and three con trol groups, and the fish in each group were cultured in a separate Inhibitors,Modulators,Libraries tank. The fish in V1 V3 groups were intramuscularly injected with 1��105 CFU fish 1 of WED bacteria in 5 ul phosphate buffered saline, as previously described, and the fish in C1 C3 groups were i. m. injected with 5 ul PBS alone. After two days of immunization, 20 fish from each of the three WED immunized and three mock immunized groups were sacrificed under anesthesia to obtain liver samples, and subsequently stored at ?80 C until RNA extraction for RNA seq analysis.

Meanwhile, 10 fish from each group were sacrificed under anesthesia at days 1, 2, 3, and Inhibitors,Modulators,Libraries 5 post immunization to obtain liver and spleen tis sue samples, and subsequently stored at ?80 C until RNA extraction for real time qPCR analysis. Inhibitors,Modulators,Libraries All the zeb rafish were handled Inhibitors,Modulators,Libraries in compliance with the local animal welfare regulations and maintained according to stand ard protocols. The immunization ex periment was approved by the animal center at the East China University of Science and Technology. Library preparation and sequencing Total RNA was extracted from each tissue sample using the TRIzol reagent according to the manufacturers instructions. To remove residual gen omic DNA, the RNA samples were incubated with 10 units of DNA free DNAse I for 30 min at 37 C.

The quality and quantity of the purified RNA were determined by measuring the absorbance at 260 nm 280 nm using a Nanodrop ND 1000 spectrophotometer. RNA integrity was further verified by electrophoresis through a 1. 5% agarose gel. Poly mRNA was isolated from the total RNA sam ples with oligo magnetic beads. The purified mRNA was fragmented by the RNA fragmenta tion kit and applied selleckchem Cisplatin as template for first strand cDNA synthesis using random hexamer primers and reverse transcriptase. The second strand cDNA was synthesized using RNase H and DNA polymerase I.