PMC:4502368 / 6044-13133 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/4502368","sourcedb":"PMC","sourceid":"4502368","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4502368","text":"Materials and Methods\nChinook salmon eggs obtained from Merced River Hatchery in November 2010 were reared at the University of California, Davis, in partially re-circulated aerated well water chilled to 12° (±1°) and fed commercial salmon feed (soft-moist formulation, 2–10% body weight; Rangen Inc.) until the time of the experiment, approximately 5 months posthatch. Fish were raised in a single 160-liter circular flow-through tank for 1 month preceding the experiment (see Figure 1 for experimental design), supplied with aerated well water and with a 12-hr light–12-hr dark photoperiod. The night prior to the experiment 55 fish were randomly assigned to one of five treatment groups (11 fish per treatment) and were allowed to acclimate at 12° in the experimental chambers overnight. Experimental chambers consisted of 5-gallon buckets with large mesh windows in the sides to allow water to freely enter and exit the chamber when submerged. Each chamber included an air stone to ensure well-aerated treatment water as well as to prevent thermal stratification. Water in the larger tanks was also well-aerated to maintain oxygen saturation. Treatments consisted of a 3-hr exposure to 15°, 18°, 21°, or 25° (±0.5°), followed by a 1-hr of recovery period at 12° (±0.5°). These temperatures range from optimal to the upper thermal limit for Chinook salmon determined by critical thermal methodology (Myrick and Cech 1998). Three hours was chosen as an ecologically relevant time exposure, because it approximates potential exposure times to warm water during juvenile outmigration and passage through warmer river reaches (Michel et al. 2013). The experimental chambers were moved to tanks held at a constant temperature using submersible titanium heating elements (Finnex 300W), facilitating very rapid change in the temperature experienced by the fish. Controls were handled identically to the other four treatment groups but remained at 12° (±0.5°). Following recovery, fish were killed with buffered tricaine methanesulfonate (500 mg/L tricaine and 420 mg/L NaCO3), weighed and measured, and gill tissue was immediately collected [there was no significant difference in weight (P = 0.53) or length (P = 0.79) of fish between groups]. Samples were preserved in RNAlater solution (Life Technologies) and stored at −80° prior to RNA extraction. Replicates of this temperature experiment were performed on 3 consecutive days at 9:00 am, yielding three replicates of 11 individuals at each temperature. Treatment of all animals was in accordance with University of California, Davis, animal care and use protocol #17875. RNA was isolated from gill tissue of 165 individuals using the TissueLyser II bead mill (Qiagen) for tissue homogenization and TRIzol Reagent Solution (Applied Biosystems) according to the manufacturer’s protocol. RNA was quantified and quality checked using the 2100 Bioanalyzer (Agilent) and RNA 6000 Nano Kit (Agilent). All RNA had a RIN (RNA Integrity Number) of 9.5 or higher. RNA from the 11 individuals in each experimental replicate was proportionally pooled and used to generate sequencing libraries using the Illumina TruSeq RNA Sample Preparation Kit and associated protocol (TruSeq RNA Sample Preparation Guide, part #15008136, November 2010 release). The 15 libraries were barcoded and processed, three libraries to a lane, with 100-bp paired-end sequencing on the Illumina HiSeq2000 platform at the University of California, Berkeley Vincent J. Coates Genomic Sequencing Laboratory. Raw data can be found at BioProject, record GSE59756.\nFigure 1 Experimental design. The resulting sequences were quality-filtered (Phred quality score cutoff = 20), adapter sequences were removed from the beginning of each read, and 20 bp were trimmed from the end of each read to remove lower-quality bases (Cutadapt version 1.1). Trimmed reads were pooled and duplicates were removed before performing the de novo reference transcriptome assembly. Assembly was performed in CLC Genome Workbench (version 4.9 beta) using default settings for de novo assembly and a minimum contig length of 100 bp. Differentially expressed (DE) transcript identification was performed using CLC Genome Workbench (version 5.0). The sequences from each treatment group were then aligned back to the reference transcriptome and DE transcripts compared with the 12° control group were identified using Baggerly’s test with an FDR of 0.10 (Baggerly et al. 2003). The threshold for significance was set at P \u003c 0.01 (FDR-corrected) and greater than two-fold change in expression. The de novo contigs were annotated with sequence descriptions identified through BLAST (Altschul et al. 1990) searches of the NCBI nucleotide database and assigned with GO terms, enzyme codes, KEGG pathways, and InterPro matches using default parameters in Blast2GO (Conesa et al. 2005).\nDirection and magnitude of expression of three genes (SERPINH1, FOS, and CXCL8) in the pooled samples were confirmed with qPCR. RNA was treated with Deoxyribonuclease I, Amplification Grade (Life Technologies), to remove possible genomic DNA contamination according to manufacturer’s instructions. DNase-treated RNA was converted to cDNA using the SuperScript VILO cDNA Synthesis Kit (Life Technologies) according to the manufacturer’s instructions. Quantitative PCR was then performed using BioRad Chromo4 real-time detector in an 8 μl reaction [1 μM forward primer, 1 μM reverse primer, 1X Quantitect SYBR Green (Qiagen), and 5 ng cDNA]. Cycling parameters were 95° (15 min), 94° (15 sec), 58° (30 sec), 72° (30 sec) (data acquisition) × 45 cycles, followed by a melt curve between 55° and 95° to ensure a single amplicon was present (Table 1). Standard curves were performed for each primer pair to ensure PCR efficiency was between 95% and 105%. Three replicates were performed for each sample and two no-template controls were included on each plate, and a no-RT control was included for each sample. Quantitative PCR was also performed for selected genes on individual RNA samples to assess expression variation within a pooled treatment group (Supporting Information, Figure S1). Quantitative PCR results were normalized to the housekeeping gene EF1A after ensuring its expression level remained stable across samples and experimental conditions. Analysis of qPCR data was performed using the delta delta Ct method, and results for all three technical replicates for a given sample were required to be within 10% of each other to be included.\nTable 1 Primer sequences and GenBank accession numbers for gene expression validated by qPCR\nGene Group Gene Name Gene Symbol GenBank Accession Number Primers (5′-3′)\nProtein folding Heat shock protein 47 SERPINHI AB196463.1 F-GTTCCCATGATGCATCGCAC\nR-CCTTGGTTTTGTCCACAGCG\nTranscription C-fos FOS AB111054.1 F-AATGACTTTGAGCCCCTGTG\nR-GTAGGGGAGCTGAGGGAATC\nInflammation/\u2028immunity Chemokine ligand 8 CXCL8 AY160982.1 F-AGAATGTCAGCCAGCCTTGT\nR-CTTGCTCAGAGTGGCAATGA\nHousekeeping Elongation factor 1A EF1A FJ890356.1 F-TCTCAGGCTGATTGCGCTGT\nR-GGGGGCTCAGTAGAGTCCAT","divisions":[{"label":"title","span":{"begin":0,"end":21}},{"label":"p","span":{"begin":22,"end":3573}},{"label":"figure","span":{"begin":3574,"end":3604}},{"label":"label","span":{"begin":3574,"end":3582}},{"label":"caption","span":{"begin":3584,"end":3604}},{"label":"p","span":{"begin":3584,"end":3604}},{"label":"p","span":{"begin":3605,"end":4865}},{"label":"p","span":{"begin":4866,"end":6515}},{"label":"label","span":{"begin":6516,"end":6523}},{"label":"caption","span":{"begin":6525,"end":6609}},{"label":"title","span":{"begin":6525,"end":6609}},{"label":"tr","span":{"begin":6610,"end":6687}},{"label":"th","span":{"begin":6610,"end":6620}},{"label":"th","span":{"begin":6622,"end":6631}},{"label":"th","span":{"begin":6633,"end":6644}},{"label":"th","span":{"begin":6646,"end":6670}},{"label":"th","span":{"begin":6672,"end":6687}},{"label":"tr","span":{"begin":6688,"end":6772}},{"label":"td","span":{"begin":6688,"end":6703}},{"label":"td","span":{"begin":6705,"end":6726}},{"label":"td","span":{"begin":6728,"end":6736}},{"label":"td","span":{"begin":6738,"end":6748}},{"label":"td","span":{"begin":6750,"end":6772}},{"label":"tr","span":{"begin":6773,"end":6795}},{"label":"td","span":{"begin":6773,"end":6795}},{"label":"tr","span":{"begin":6796,"end":6857}},{"label":"td","span":{"begin":6796,"end":6809}},{"label":"td","span":{"begin":6811,"end":6816}},{"label":"td","span":{"begin":6818,"end":6821}},{"label":"td","span":{"begin":6823,"end":6833}},{"label":"td","span":{"begin":6835,"end":6857}},{"label":"tr","span":{"begin":6858,"end":6880}},{"label":"td","span":{"begin":6858,"end":6880}},{"label":"tr","span":{"begin":6881,"end":6966}},{"label":"td","span":{"begin":6881,"end":6903}},{"label":"td","span":{"begin":6905,"end":6923}},{"label":"td","span":{"begin":6925,"end":6930}},{"label":"td","span":{"begin":6932,"end":6942}},{"label":"td","span":{"begin":6944,"end":6966}},{"label":"tr","span":{"begin":6967,"end":6989}},{"label":"td","span":{"begin":6967,"end":6989}},{"label":"tr","span":{"begin":6990,"end":7066}},{"label":"td","span":{"begin":6990,"end":7002}},{"label":"td","span":{"begin":7004,"end":7024}},{"label":"td","span":{"begin":7026,"end":7030}},{"label":"td","span":{"begin":7032,"end":7042}},{"label":"td","span":{"begin":7044,"end":7066}}],"tracks":[{"project":"2_test","denotations":[{"id":"25911227-12912827-43383293","span":{"begin":4456,"end":4460},"obj":"12912827"},{"id":"25911227-2231712-43383294","span":{"begin":4682,"end":4686},"obj":"2231712"},{"id":"25911227-16081474-43383295","span":{"begin":4859,"end":4863},"obj":"16081474"}],"attributes":[{"subj":"25911227-12912827-43383293","pred":"source","obj":"2_test"},{"subj":"25911227-2231712-43383294","pred":"source","obj":"2_test"},{"subj":"25911227-16081474-43383295","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec93e8","default":true}]}]}}