|
| Status |
Public on Jan 23, 2018 |
| Title |
15690 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
3-WTD3
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Columbia 0
genotype: wild type
age: 4 days
tissue: seedling
replicate: 3
treatment: darkness
|
| Growth protocol |
Seedlings were grown on half-strength MS medium without sucrose. 48h 4°C, 4 days in darkness 22°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
QIAGEN RNeasy Plant Mini Kit with on-column DNase digestion and four RPE washes.
|
| Label |
Cy5
|
| Label protocol |
RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies), and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Per sample, an amount of 200 ng of total RNA spiked with 10 viral polyA transcript controls (Agilent) was converted to double-stranded cDNA in a reverse transcription reaction. Subsequently, the sample was converted to antisense cRNA, amplified and labeled with Cyanine 5-CTP (Cy5) in an in vitro transcription reaction according to the manufacturer's protocol (Agilent).
|
| |
|
| Channel 2 |
| Source name |
11-WTFR3
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Columbia 0
genotype: wild type
age: 4 days
tissue: seedling
replicate: 3
treatment: far-red light
|
| Growth protocol |
Seedlings were grown on half-strength MS medium without sucrose. 48h 4°C, 4 days in darkness 22°C, 1h far-red light.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
QIAGEN RNeasy Plant Mini Kit with on-column DNase digestion and four RPE washes.
|
| Label |
Cy3
|
| Label protocol |
RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies), and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Per sample, an amount of 200 ng of total RNA spiked with 10 viral polyA transcript controls (Agilent) was converted to double-stranded cDNA in a reverse transcription reaction. Subsequently, the sample was converted to antisense cRNA, amplified and labeled with Cyanine 3-CTP (Cy3) in an in vitro transcription reaction according to the manufacturer's protocol (Agilent).
|
| |
|
| |
| Hybridization protocol |
A mixture of purified and labeled cRNA (Cy3 label: 825 ng; Cy5 label: 825 ng) was hybridised on the Agilent-021169 Arabidopsis 4 Oligo Microarray (V4) followed by (manual) washing, according to the manufacturer's procedures.
|
| Scan protocol |
To assess the raw probe signal intensities, arrays were scanned using the Agilent DNA MicroArray Scanner with SureScan High-Resolution Technology, and probe signals were quantified using Agilent's Feature Extraction software (version 10.7.3.1).
|
| Data processing |
We use the Agilent processed signal values (i.e., feature gProcessedSignal for the Cy3 signal and rProcessedSignal for the Cy5 signal) from Agilent Feature Extraction software v10.7.3.1 and compute log2-(Cy5/Cy3)-ratios. We remove Agilent control probes, as well as probes with signals below background on all arrays (absent spots). To decide whether a signal is significantly above background, we use the features gIsPosAndSignif and rIsPosAndSignif, also provided by the Agilent software. In case of multiple probes for the same Agilent ID, log2-(Cy5/Cy3)-ratios are averaged.
|
| |
|
| Submission date |
Nov 06, 2012 |
| Last update date |
Jan 24, 2018 |
| Contact name |
Rekin's Janky |
| E-mail(s) |
[email protected]
|
| Organization name |
VIB
|
| Department |
Nucleomics Core
|
| Street address |
Herestraat 49 Box 816
|
| City |
Leuven |
| ZIP/Postal code |
B-3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL12621 |
| Series (1) |
| GSE42053 |
Plant Elongator in light signaling |
|
