|
| Status |
Public on Dec 13, 2012 |
| Title |
RP1178-B3-48H-27C |
| Sample type |
RNA |
| |
|
| Source name |
Acropora palmata larval culture
|
| Organism |
Acropora palmata |
| Characteristics |
tissue type: larval culture
batch: batch 3
age: 48 hours
temperature: 27°C
|
| Treatment protocol |
Larvae were reared at a control temperature (27°C) or elevated temperature (29°C). Replicates of each batch culture (n = 2) were kept in separate 1L plastic containers (with mesh sides to allow for water exchange). Plastic 1 L containers (n = 10) were suspended in replicate 45L polycarbonate bins (n = 4) at each temperature (n = 2). Water in each bin was circulated with an aquarium pump and changed daily with filtered sea water preheated to the target temperature. Temperatures were maintained within ±1°C with ¼ hp aquarium chillers (Current USA, CA) and monitored with HOBO data loggers (Onset Co., MA).
|
| Growth protocol |
Gametes from multiple parent colonies were collected during the 2009 mass spawning event on the reef off of Rincon, Puerto Rico. The genotypic identity of each colony targeted for gamete collection was determined using microsatellite markers (Baums, Hughes et al. 2005). Batch cultures, consisting of four genotypically distinct parents each, were generated by combining sperm and eggs from selected parent colonies. After fertilization (one hour), the zygotes were rinsed with filtered sea water and transferred to temperature controlled aquaria.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Embryos for the microarray experiment were sampled at 24, 48, and 72 hours (only samples from batch 2 were available at 72h) and preserved in RNAlater (Ambion, TX) followed by storage at -80°C. Total RNA was extracted from approximately 30-100 larvae from each sample using the RNeasy Mini Kit (Qiagen, CA). Quality and concentration of total RNA was as-sessed on an Agilent 2100 Bioanalyzer 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 hy-bridized 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 manufactur-er’s instructions, then dried down completely in a speed-vac.
|
| |
|
| 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 |
This sample was not used for analysis after initial normalization. A batch extract from 30-100 coral larvae derived from the same set of four parent colonies from Rincon, Puerto Rico, and raised at a common temperature for a specified time since fertilization.
|
| 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 Aquantile 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 |
Apr 02, 2012 |
| Last update date |
Dec 13, 2012 |
| Contact name |
Nicholas Polato |
| E-mail(s) |
[email protected]
|
| 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) |
| GSE36983 |
Variation in the transcriptional response of threatened coral larvae to elevated temperatures |
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