|
| Status |
Public on Oct 01, 2015 |
| Title |
Dynamic1_Hot34C_GenotypeB_MR297 |
| Sample type |
RNA |
| |
|
| Source name |
Acropora palmata adult tissue homogenate
|
| Organism |
Acropora palmata |
| Characteristics |
phenotype: Dynamic
phenotype replicate: 1
temperature: 34°C
temperature class: Hot
genotype: B
sample id: MR297
|
| Treatment protocol |
Fragments (~10 cm2) from each of the target coral genotypes were transported to temperature-controlled aquaria. One piece of each genotype was transferred to one of three separate bins containing filtered seawater maintained at temperatures of 20 (cold), 27 (ambient), or 34°C (hot). The fragments were kept in temperature treatments for three days; temperatures stayed within ±0.4°C of the targets.
|
| Growth protocol |
In Spring 2011, colonies of Acropora palmata were sampled from a reef in Puerto Morelos, Mexico (N 20°52.461', W 86°51.073') and genotyped at five neutral microsatellite loci according to Baums et al. (2005). The Symbiodinium ‘fitti’ strain in each colony was genotyped at ten microsatellite loci (Pinzon et al. 2011). Six of the host genotypes harbored the same strain of S ‘fitti’. These six colonies were targeted for temperature experiments. Colonies hosting symbionts with a small cold stress response (hosts B and Z) and a large cold stress response (hosts D and Y) were binned as replicates. Their host gene expression was contrasted via microarray. Each holobiont phenotype was subsequently called Dynamic and Static, respectively.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the ~3 polyps of each host genotype at each temperature taken 3.5 h after exposure to treatment. Extractions were performed at the Pennsylvania State University with the RNeasy Mini Kit (Qiagen, CA). Concentration and quality of RNA extracts were quantified on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) to ensure that high molecular weight RNA was present.
|
| Label |
Cy5
|
| Label protocol |
To prepare samples for microarray hybridization, one cycle of amplification was per-formed on 1ug of each RNA sample using the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion Life Technologies, AM1753) following the manufacturer’s protocol. Dye coupling of 15 ug of aRNA was performed with Cy3 or Cy5 (GE Health Care, RPN5661), and subsequently purified according to the Ambion Kit instructions. For each pair of samples that were to be hybridized to the same array, 2µg each of the Cy3 and Cy5 labeled sample were combined and fragmented using RNA Fragmentation Reagents (Ambion, AM8740) according the manufacturer’s instructions, then dried down completely in a speed-vac
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| |
|
| Hybridization protocol |
Samples were resuspended in the appropriate tracking control and hybridization solution master mix was added following manufacturer’s instructions (Roche NimbleGen). Following two 5-minute incubations at 95°C and 42°C, the mixer was attached to the array and hybridization solutions were added according to the manufacturer’s instructions. Hybridization was performed while mixing overnight at 42°C in a MAUI hybridization instrument (BioMicro Systems, UT). Hybridized slides were washed according to the manufacturer’s instructions (Roche NimbleGen), and spun at 1000 RPM for 3 minutes in a 50 ml conical tube filled with N2 gas. Dried slides were placed in fresh tubes with 2.5 ml of DyeSaver (Genisphere Inc.) and rotated for 45 seconds to coat the slide. A final spin at 700 RPM for 3 seconds removed excess DyeSaver.
|
| Scan protocol |
Scanning was performed with an Axon GenePix 4000B
|
| Description |
Both host and symbiont RNA are present in sample.
|
| Data processing |
Raw probe intensities were read into R for analysis in the Bioconductor package LIMMA (Smyth 2005; R Development Core Team 2008). Probe intensities were normalized within arrays using lowess normalization, and between arrays using the quantile normalization method which ensures that the average intensities have the same empirical distribution across arrays (Yang and Thorne 2003).
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| |
|
| Submission date |
Sep 17, 2013 |
| Last update date |
Oct 01, 2015 |
| Contact name |
John Everett Parkinson |
| E-mail(s) |
jparkinson@psu.edu
|
| Organization name |
Penn State University
|
| Department |
Biology
|
| Lab |
Baums
|
| Street address |
208 Mueller Lab
|
| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
| |
|
| Platform ID |
GPL15393 |
| Series (1) |
| GSE50926 |
Intraspecific diversity among partners drives functional variation in coral symbioses |
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