The DDB2 cDNA was also subcloned into a pEF1/Myc-HisB vector between the KpnI and XbaI sites, to produce a Myc-polyhistidine-tagged DDB2 protein

Muscle sequencing, a high-throughput transcriptomic approach, to provide an in-depth view of the transcriptome of red and white skeletal muscle in rainbow trout. To the best of our knowledge, our study represents the first application of RNA-seq to the study of the skeletal muscle transcriptome in rainbow trout. We have chosen to use rainbow trout because it is an economically important species for aquaculture and because it displays migrant behaviour. Still, in absence of a reference genome of rainbow trout, the nucleotide transcripts need to be de novo assembled to larger groups of sequences representing 25833960 overlapping regions from the transcriptome. Contigs can then be annotated using iterative sequence homology searches against known related sequences such as salmonid ESTs or the zebrafish genome. In the present study, we used RNA-seq to catalogue the red and white muscle transcriptome in rainbow trout. We also aimed to investigate the effects of exercise in red and white muscle and, specifically, to focus on muscle building versus muscle wasting processes and on the potential interaction between skeletal muscle and the reproductive axis. The gained information should allow us to determine whether the rainbow trout muscle matched the migration phenotype or the sexual maturation phenotype after the long-term exercise of simulated reproductive migration. Materials and Methods The Experimental Set-up The swimming MedChemExpress Cilomilast experiment was performed in a 6,000 L swimflume recently described in Palstra et al.. In short, an oval shaped swim-flume had been placed in a 100 m2 climatized room. In one of the straight ends of the swim-flume, a compartment of 2.060.7 m was created with two mesh fences. This compartment was divided by a PVC fence that started in the curve. The resulting inner compartment, where the current was null, was used to house the resting group. The outer compartment where the current was maximal was used to house the swimming group. Thus, both groups were subjected to identical conditions except for swimming exercise. Initially, water in the swim-flume was brackish at 10 %, created by mixing natural seawater from the Oosterschelde at the Burgersluis as delivered by truck, with tap water. Flow was created and speed profiles were measured as described before as well as water temperature and water quality parameters that were monitored and controlled. 16 h directly to the swim-flume at Leiden University. Fish were randomly divided into a `rest’-group and a `swim’-group. During the following 4 days, the brackish 10 % water was stepwise replaced by freshwater at 16uC and photoperiod was changed from 16L:8D to 8L:16D. Fish were then acclimatized for two days to their new conditions. As in our previous study, swimmers were first swum at a speed of 0.33 body-lengths per second that was increased the next day to the final, near optimal speed of 0.75 BL/s and all fish were sampled after 40 days. Fish did not show any signs of fatigue, stress or health problems during 40 days of resting or swimming without feeding. After 40 days, a distance of 1,176 km had been swum continuously in a sustained manner. At sampling, fish were anesthetized using oil 23838678 of cloves, euthanized by decapitation and dissected for red and white muscle tissue from standardized locations. Samples were flash frozen in liquid nitrogen and stored at 280uC for RNA sequencing and Q-PCR. In addition, the gonadosomatic index was measured from trout at the beginning of the experiment

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