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| Status |
Public on Nov 22, 2017 |
| Title |
2 µM TDCIPP-exposed embryo at 6 hpf, Biological rep-1, Technical rep-2 |
| Sample type |
RNA |
| |
|
| Source name |
Zebrafish embryos exposed to 2 µM TDCIPP from 0.75 to 6 hpf
|
| Organism |
Danio rerio |
| Characteristics |
tissue: whole embryo
agent: TDCIPP
time: 0.75 to 6 hpf
|
| Treatment protocol |
Treated with 0.1% DMSO (Control) or 2 µM TDCIPP (Treatment)
|
| Growth protocol |
Embryos were collected and sorted based on their stages, exposed to treatment solutions in glass beakers at 0.75 hpf (2-cell stage) and incubated at 28°C under artificial light till 2 or 6 hpf.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from pooled embryos using a SV Total RNA Isolation System (Promega). After elution of RNA in 100 μL nuclease-free water, total RNA concentrations, 260/280 ratios, and 260/230 ratios were quantified using a NanoDrop ND-2000 spectrophotometer (ThermoFisher Scientific) and then stored at -80°C; total RNA quality was also confirmed using an Agilent 2100 Bioanalyzer system.
|
| Label |
biotin
|
| Label protocol |
Total RNA samples were amplified and biotinylated using GeneChip WT PLUS Reagent Kit (Affymetrix). Briefly, 100 ng of total RNA per sample was reverse-transcribed into ds-cDNA using NNN random primers containing a T7 RNA polymerase promoter sequence; cDNA quality was then confirmed using an Agilent 2100 Bioanalyzer system. T7 RNA polymerase was then added to cDNA samples to amplify RNA, and then RNA was reverse-transcribed to ss-DNA and degraded using RNase H. ss-DNA molecules were then fragmented and terminally labelled with biotin.
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| |
|
| Hybridization protocol |
Amplified and labeled samples were hybridized onto duplicate Zebrafish Gene 1.0 ST Arrays (Affymetrix) for 16 h at 45°C using a GeneChip Hybridization Oven 640 and a GeneChip Hybridization, Wash, and Stain Kit (Affymetrix); these arrays were constructed by Affymetrix based on the danRer6/Zv9 genome build and contained 1,255,682 probes representing 59,302 unique genes.
|
| Scan protocol |
Arrays (24 total) were then scanned using a GeneChip Scanner 3000 7G system and computer workstation equipped with GeneChip Command Console 4.0 software (Affymetrix).
|
| Data processing |
Following completion of array scans, probe cell intensity (CEL) files (24 total) were imported into Expression Console Software (Affymetrix) and processed at the gene-level using Affymetrix’s ZebGene-1_0-st library file and Robust Multichip Analysis (RMA) algorithm to generate CHP files. After confirming data quality within Expression Console, CHP files containing log2 expression signals for each probe were then imported into Transcriptome Analysis Console Software (Affymetrix) and R (www.r-project.org) to analyze treatment-specific transcriptional responses using volcano plots and one-way between-subject analysis of variance (ANOVA). Affymetrix transcript cluster IDs for differentially expressed genes with False Discovery Rates (FDR) of p < 0.1 were imported into DAVID Bioinformatics Resources 6.8 for Gene Ontology (GO) enrichment analysis against zebrafish genome assembly GRCz10.
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| |
|
| Submission date |
Nov 14, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
David Volz |
| E-mail(s) |
david.volz@ucr.edu
|
| Organization name |
University of California, Riverside
|
| Department |
Environmental Sciences
|
| Street address |
900 University Ave.
|
| City |
Riverside |
| State/province |
CA |
| ZIP/Postal code |
92521 |
| Country |
USA |
| |
|
| Platform ID |
GPL16933 |
| Series (1) |
| GSE106875 |
Microarray-based transcriptomic responses of zebrafish embryos exposed to 2 µM TDCIPP from 0.75 hpf to 2 and 6 hpf |
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