|
| Status |
Public on Mar 22, 2016 |
| Title |
WT_4 |
| Sample type |
RNA |
| |
|
| Source name |
Primary tissue_WT_4dpf_30 embryos
|
| Organism |
Danio rerio |
| Characteristics |
strain: TL/LWT mix
genotype/variation: wild type
age: 4 days post fertilisation
tissue: Primary tissue from 30 embryos
|
| Growth protocol |
Embryos were for 4 days incubated at 28oC in E3 (5mM NaCl, 0.17mM KCl, 0.33mM CaCl2, 0.33mM MgCl2, pH 7.2) medium containing 0.0001 % methylene blue (Sigma-Aldrich).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Extraction of total RNA from zebrafish embryos was performed using the mIRVANA miRNA isolation kit (Invitrogen) excluding the step for enrichment of miRNA. This kit uses a glass fiber filter (GFF)-based method, to reduce size bias. Extracted RNA was quantified using the Nanodrop ND-1000 spectrophotometer. Quality of RNA was assessed using Agilent Bioanalyser 2100 Nanochip 6000, samples with RNA Integrity Numbers ≥ 7 were accepted.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 400ng RNA using the One-Color Microarray- One-Colour Input Quick Amp Labelling kit version 5.7 (Agilent) according to manufacturer's instructions, followed by RNeasy mini spin columns (Qiagen).
|
| |
|
| Hybridization protocol |
1.65 µg of Cy-3-labelled cRNA (specific activity > 9.0 pmol Cy3 per µg cRNA) was fragmented at 60°C for 30 minutes in reaction volume of 30 µL containing 1 X Agilent fragmentation buffer and 2X Agilent blocking agent following the manufacturer's instructions. To terminate fragmentation, 30 µL 2x GEx Hybridization Buffer HI-RPM was added to the reaction. The sample was hybridized to custom-designed whole genome zebrafish oligo microarrays (G2514F) for 17 hours at 65oC in a rotating Agilent hybridization oven. After hybridization, microarrays were washed for 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37oC GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
GenePix 4000B (Axon Instruments) according to manufacturer’s protocol (One-Colour Microarray-Based Gene Expression Analysis (Quick Amp Labelling), Version 5.7, March 2008). The scanner was set to 4X44K array slides (Scan area 61 x 21.6 mm, Scan resolution 5µm, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression in wild-type at 4dpf
|
| Data processing |
Agilent Feature Extraction Software version 10.7.3.1 (standard protocol GE1_107_Sep09) was used for data extraction, background correction and flagging of non-uniform features. As we used custom arrays, the output of Feature Extraction was processed using the GIS Unigene & Gene Ontology Annotation Tool (http://123.136.65.67/) to find Genbank Accession and Unigene ID’s using Danio rerio Build (DR Build #126_66). Limma Version 3.24.10 (Biocondutor). Background correction was applied using the “normexp” function and normalisation was performed using normalise between arrays “quantile” function. To average replicate spots, the “avereps” function was applied. The linear model was applied using “lmfit” and a contrast matrix was applied using eBayes (fit2). From these normalised entity lists, a significance cut off of p≤ 0.01 and fold change cut off of ≥ 2 were applied.
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| |
|
| Submission date |
Jul 14, 2015 |
| Last update date |
Mar 22, 2016 |
| Contact name |
Justin Jeyakani Joseph Gnanakkan |
| E-mail(s) |
gnanakkan@gis.a-star.edu.sg
|
| Phone |
68088173
|
| Organization name |
Genome Institute of Singapore
|
| Department |
Computational and Systems Biology
|
| Street address |
60, Biopolis street
|
| City |
Singapore |
| State/province |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL10816 |
| Series (2) |
| GSE70885 |
Genome-wide mapping of Hif-1α binding sites in zebrafish [microarray] |
| GSE70886 |
Genome-wide mapping of Hif-1α binding sites in zebrafish |
|
