title: Illumina HiSeq 2000 paired end sequencing; Transcriptome-wide mapping of signaling pathways and early immune responses in lumpfish leukocytes upon in vitro bacterial exposure
design: Transcriptome-wide mapping of signaling pathways and early immune responses in lumpfish leukocytes upon in vitro bacterial exposure
library_construction_protocol: Farmed lumpfish (C. lumpus L.) were provided from Fjord Forsk Sogn AS, a commercial breeder in Sogn & Fjordane County, Norway. The fish were kept in a 500 L tank at the Aquatic and Industrial Laboratory (ILAB) within the High-Technology Centre in Bergen under normal rearing conditions with a light regime 12h light: 12h dark. The water temperature was 8ºC, salinity 34 PSU and a minimum of 77% oxygen in the outlet water. The fish were fed with the commercial dry feed Amber Neptune (3 mm). Head kidney leukocytes were isolated as described previously using discontinuous Percoll gradients 49. Both left and right kidney lobes from 15 fish were included. Cell number, viability and aggregation factor was determined using a CASY Cell Counter™ (Innovatis AG). For in vitro bacterial exposure, 5 x 106 cells in L-15+ medium without antibiotics were added to each well in a 24-well plate (Nunc) and mixed with the bacteria Vibrio anguillarum O1 (MOI 1:10) in a total volume of 0.5 mL. In wells with non-exposed cells, medium was added instead of bacteria. The plates were incubated at 15ºC. After 1.5 hour, pencillin/streptomycin was added to each well and the plates were further incubated until 6hrs and 24 hours post exposed. In order to obtain an as comprehensive transcriptome as possible, a sample with leukocytes exposed with infectious pancreatic necrosis virus for 24 hrs was also included The bacteria was grown in tryptic soy broth containing 2.0% NaCl, at 20 degrees celcius and at 200 revolutions per minute. The bacteria were harvested in late exponential phase. 5x10^6 head kidney leukocytes in L-15+ medium without antibiotics were added to each well in a 24-well plate (Nunc) and mixed with the bacteria Vibrio anguillarum O1 (5*10^7 bacteria) in a total volume of 0.5 mL. In wells with non-exposed cells, medium was added instead of bacteria. The plates were incubated at 15ºC. After 1.5 hour, pencillin/streptomycin was added to each well and the plates were further incubated until 6hrs and 24 hours post exposed. Total RNA was isolated using GeneElute Mammalian Total RNA miniprep kit (Sigma) according to the manufacturer's instructions. Samples were treated with DNase I (Sigma) to removed traces of genomic DNA and the concentration of total RNA determined in a Nanodrop®ND-1000 UV-Vis spectrophotometer (Nanodrop Technologies). Total RNA extracts from five fish were pooled, 1000 ng from each fish, making three parallels from each time point in the de novo RNA sequencing. The pooled RNA (5 µg) was cleaned using RNA clean & concentrator-5 (zymo research) according to the manufacturer's instructions and the quality of the RNA were determined in an Agilent 2100 bioanalyzer. RNA isolated from virus infected leukocytes was kept separately. The RQI values were in the range 6.3-9.3. The Norwegian High Throughput Sequencing Centre prepared sequencing libraries using TruSeq™RNA sample Preparation kit (Illumina®) according to the manufacturer's protocol
attributes.tag: Experimental Factor: infect
attributes.value: none
attributes.tag: Experimental Factor: time
attributes.value: 24