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Status |
Public on Aug 30, 2018 |
Title |
Intestine_zebrafishfood_zebrafishbiome_rep4 |
Sample type |
RNA |
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Source name |
intestine
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Organism |
Danio rerio |
Characteristics |
treatment: antibiotic treated (7 days), zebrafish biome replaced, fed zebrafish food (14 days) tissue: intestine gender: female age: Adult
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Treatment protocol |
Zebrafish were treated with anitbiotics (except wild types) for 7 days. The water was changed daily and fresh anitbiotics added. The temperature and day:night cycle were maintained. After 7 days, the water was changed and animals allowed to recover for 1 day. Subsequently, animals were implanted with either zebrafish microbiomes or goldfish microbiomes. The animals were then fed daily either zebrafish food or goldfish food for 14 days. The five treatment groups were housed seperatley to prevent contamination.
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Growth protocol |
Prior to experimentation all zebrafish were housed in aquaria with reciculating water kept at 25 C and a 12h:12h day:night cycle. Goldfish were kept in a separate reciculating system at 25 C and a 12h:12h day:night cycle. All animals were fed daily.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted from frozen intestinal samples using Trizol reagent following the manufacturer’s instructions (Invitrogen). Purity and integrity of the extracted RNA was validated on a 2100 Bioanalyzer Instrument (Agilent Technologies)
|
Label |
Cy3
|
Label protocol |
A total of 500 ng of total RNA was labeled following the explicit instructions of the one-color microarray based gene expression analysis with low input quick Amp labeling protocol (Agilent Technologies).
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Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
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Scan protocol |
Slides were scanned immediately after washing on SureScan Microarray Scanner G2600D using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
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Description |
Gene expression 14 days after antibiotic treatment, biome replacement, and no diet change in D. rerio intestine
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Data processing |
Raw fluorescent data files were converted to normalized expression indices using the RMAalgorithm in GeneSpring GX (Agilent Technologies).
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Submission date |
Aug 13, 2018 |
Last update date |
Aug 30, 2018 |
Contact name |
Paul Craig |
E-mail(s) |
pcraig@uwaterloo.ca
|
Organization name |
University of Waterloo
|
Department |
Biology
|
Street address |
200 University Ave West
|
City |
Waterloo |
State/province |
ON |
ZIP/Postal code |
N2L3G1 |
Country |
Canada |
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Platform ID |
GPL14688 |
Series (1) |
GSE118496 |
Fecal transplants in zebrafish reveal effects of diet and microbiome on intestinal gene expression and enzyme activity. |
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