|
| Status |
Public on Sep 18, 2017 |
| Title |
Treatment 1 conditions 1 vs 2 replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
treatment 1, condition 2
|
| Organism |
Daphnia pulex |
| Characteristics |
condition: heat shock to 30C
treatment: 2hr
|
| Treatment protocol |
Time-resolved experiments (acute heat stress) were carried out with long-term 20°C acclimated animals, which were exposed for three different time intervals (2, 4, and 8 h) to either 30 ± 0.2°C (test conditions) or 20 ± 0.3°C (control conditions). For this, 50 animals each were rapidly transferred with a sieve (mesh size: 1.2 mm) from 20°C-medium to a set of beakers containing 30°C-medium. As control, 50 animals each were transferred from 20°C-medium to another set of beakers with 20°C-medium. Only adult female animals with a body length of 2−2.5 mm (between the base of the apical spine and the anterior part of the head) and carrying parthenogenetic eggs and embryos were used for experiments. Animals were not fed during short-term exposures. During experiments, all animals were in good physical condition. After heat exposure, animals were transferred into 1.5-ml microcentrifuge tubes and shock-frozen in liquid nitrogen after removing adhering water with a tissue paper. Samples were short-term stored at -80°C.
|
| Growth protocol |
The daphniids (50 adult animals per batch) were raised in 3-L glass beakers under a 16 h:8 h L:D photoperiod. Three-quarter of the culture medium (M4) was renewed once a week, and the animals were equally fed ad libitum with algae (Desmodesmus subspicatus; SAG 53.80, Göttingen, Germany). Only adult female animals with a body length of 2−2.5 mm (between the base of the apical spine and the anterior part of the head) and carrying parthenogenetic eggs and embryos were used for experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue was homogenized using Trizol reagent (Invitrogen) and extracted RNA was purified using the Qiagen RNeasy protocol with on-column DNase treatment from specific tissues. Total RNA amplified using MessageAmp II aRNA Kit (Ambion). aRNA converted to ds cDNA using SuperScript cDNA Synthesis Kit (Invitrogen).
|
| Label |
Cy3
|
| Label protocol |
DNA labeling using 1 O.D. CY-labeled random nonomer primer and 100U Klenow fragment (3>5 exo) per 1 μg double-stranded cDNA.
|
| |
|
| Channel 2 |
| Source name |
treatment 1, condition 1
|
| Organism |
Daphnia pulex |
| Characteristics |
condition: 20C
treatment: 2hr
|
| Treatment protocol |
Time-resolved experiments (acute heat stress) were carried out with long-term 20°C acclimated animals, which were exposed for three different time intervals (2, 4, and 8 h) to either 30 ± 0.2°C (test conditions) or 20 ± 0.3°C (control conditions). For this, 50 animals each were rapidly transferred with a sieve (mesh size: 1.2 mm) from 20°C-medium to a set of beakers containing 30°C-medium. As control, 50 animals each were transferred from 20°C-medium to another set of beakers with 20°C-medium. Only adult female animals with a body length of 2−2.5 mm (between the base of the apical spine and the anterior part of the head) and carrying parthenogenetic eggs and embryos were used for experiments. Animals were not fed during short-term exposures. During experiments, all animals were in good physical condition. After heat exposure, animals were transferred into 1.5-ml microcentrifuge tubes and shock-frozen in liquid nitrogen after removing adhering water with a tissue paper. Samples were short-term stored at -80°C.
|
| Growth protocol |
The daphniids (50 adult animals per batch) were raised in 3-L glass beakers under a 16 h:8 h L:D photoperiod. Three-quarter of the culture medium (M4) was renewed once a week, and the animals were equally fed ad libitum with algae (Desmodesmus subspicatus; SAG 53.80, Göttingen, Germany). Only adult female animals with a body length of 2−2.5 mm (between the base of the apical spine and the anterior part of the head) and carrying parthenogenetic eggs and embryos were used for experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue was homogenized using Trizol reagent (Invitrogen) and extracted RNA was purified using the Qiagen RNeasy protocol with on-column DNase treatment from specific tissues. Total RNA amplified using MessageAmp II aRNA Kit (Ambion). aRNA converted to ds cDNA using SuperScript cDNA Synthesis Kit (Invitrogen).
|
| Label |
Cy5
|
| Label protocol |
DNA labeling using 1 O.D. CY-labeled random nonomer primer and 100U Klenow fragment (3>5 exo) per 1 μg double-stranded cDNA.
|
| |
|
| |
| Hybridization protocol |
Labeled products were pooled to serve as a Master Mix for Array set. The products were dried in a speedvac then resuspended in water prior to hybridization, which was performed with the NimbleGen hybridization kit. A single hybrization master for a given source and biological replicate was applied to each of 12 subarrays with the appropriate Sample Tracking Control (STC) following protocols described in Lopez, J.A. and J.K. Colbourne. 2011. Dual-Labeled Expression Microarray Protocol for High-Throughput Genomic Investigations. CGB Technical Report 2011-02. doi:10.2506/cgbtr-201102.
|
| Scan protocol |
Axon GenePix 4200 Professional, 5 micron resolution
|
| Data processing |
Raw signal intensity extract with NimbleScan 2.6 Software (Roche NimbleGen) in PAIR files. Quantile normalization to convert raw to processed data.
The Sample*.txt files are the normalized data values for the probes on the array.
GEO2_Matrix.txt is the final, processed data file reporting log2 ratios of the first condition over second condition, quantile normalized, four biological replicates averaged.
|
| |
|
| Submission date |
Dec 08, 2016 |
| Last update date |
Sep 18, 2017 |
| Contact name |
John Kenneth Colbourne |
| E-mail(s) |
J.K.Colbourne@bham.ac.uk
|
| Phone |
+44 121 414 5423
|
| Organization name |
University of Birmingham
|
| Department |
School of Biosciences
|
| Lab |
Environmental Genomics Group
|
| Street address |
Edgbaston
|
| City |
Birmingham |
| State/province |
West Midlands |
| ZIP/Postal code |
B15 2TT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL11278 |
| Series (2) |
| GSE91030 |
Phenotypic plasticity of temperature induced Daphnia gene expression; experiment 2 |
| GSE91031 |
Phenotypic plasticity of temperature induced Daphnia gene expression |
|
