|
| Status |
Public on Jun 23, 2018 |
| Title |
Embryo at early neurula, injected with 0.5ng Gam1 mRNA, biological replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
Xenopus embryos 16.5 hours post fertilization, injected with 0.5ng Gam1 mRNA
|
| Organism |
Xenopus laevis |
| Characteristics |
tissue: Whole embryo
developmental stage: Early neurula
age: 16.5 hours post fertilization
treatment: Gam1 mRNA
|
| Treatment protocol |
Embryos were injected in the animal hemisphere (volumes ranged between 5 to 30 nl) and allowed to develop at room temperature in 1/3 MMR.
|
| Growth protocol |
Female X. laevis were injected with 500 units of human chorionic gonadotropin (HCG) at least 12 hours prior to spawning. Testes were isolated and stored at 4oC in high salt MBS (0.7 mM CaCl2, 108 mM NaCl, 1 mM KCl, 1 mM MgSO4, 5 mM HEPES, pH 7.8, 2.5 mM NaHCO3 ) for up to a week. Minced testis and eggs were mixed together with 3 mL 1/3 MMR (0.1 M NaCl, 2.0 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 5 mM HEPES, pH7.8) and left to fertilize at room temperature. After 10 minutes, eggs were flooded with additional 1/3 MMR and after an additional 30 minutes eggs were washed with 2% cysteine (< 5 minutes) in order to remove the jelly coating, upon which eggs were washed 8 times with 1/3 MMR.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Twenty embryos from each time point were homogenized in 500 uL proteinase K buffer (100 mM Tris pH 8, 150 mM NaCl, 12.5 mM EDTA pH 8.0, 1% SDS, 200 ug proteinase K) and incubated at 37oC for one hour. Samples were then extracted twice with an equal volume of phenol, pH 4.5, and twice with 24:1 chloroform:isoamylalcohol. RNA was purified using an RNeasy spin column (Qiagen, #74104) and precipitated with 2.5 volumes of ice-cold ethanol.
|
| Label |
biotin
|
| Label protocol |
Total RNA (50-500 ng) was used for cDNA synthesis according to the GeneChip 3’ IVT Express Kit for Affymetrix microarray chips. The cDNA was then converted to double-stranded DNA and used as a template for transcription of amplified RNA (aRNA) which was labeled with a biotin-conjugated nucleotide. aRNA samples (15 ug) were subsequently purified and fragmented to produce probes for hybridization onto Xenopus laevis 2.0 GeneChip 3’expression arrays.
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| |
|
| Hybridization protocol |
A hybridization cocktail is prepared, including the fragmented target, and probe array controls. It is then hybridized to the probe array during a 16-hour incubation, per manufacturer's recommendations (http://www.affymetrix.com/support/technical/byproduct.affx?product=X_laevis_2).
|
| Scan protocol |
The hybridized probe array is scanned by the GeneChipTM Scanner 3000.
|
| Description |
EN_Gam1.03
Gene expression data from Xenopus laevis embryos at the early neurula developmental stage which were injected at one cell with 0.5ng Gam1 mRNA.
|
| Data processing |
The Affymetrix data files were processed using Bioconductor software package (http://www.bioconductor.org/). The mean fluorescence intensity was derived from a log2 transformation of the normalized data.
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| |
|
| Submission date |
Jun 22, 2018 |
| Last update date |
Jun 23, 2018 |
| Contact name |
Paul Huber |
| E-mail(s) |
phuber@nd.edu
|
| Organization name |
University of Notre Dame
|
| Street address |
University of Notre Dame
|
| City |
Notre Dame |
| State/province |
IN |
| ZIP/Postal code |
46556 |
| Country |
USA |
| |
|
| Platform ID |
GPL10756 |
| Series (1) |
| GSE116164 |
Deficiency in SUMOylation, by injection of Gam1 mRNA into one cell embryos, leads to differential expression of genes in Xenopus |
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