|
| Status |
Public on Jun 30, 2016 |
| Title |
Col after 3h chilling treatment rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Col WT, 4 degree for 3 h, replicate 2
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: leaf
genotype/variation: wildtype
ecotype: Col
|
| Treatment protocol |
Two-week-old seedlings were transferred to 4 degree for 0, 3, and 48 h. Plant materials were then collected for RNA extraction.
|
| Growth protocol |
Arabidopsis seeds of RDM4 overexpression lines and Col WT were sown in MS plates and grown at 23/18°C in a growth room with a 16-h light/8-h dark cycle at 150 μmol m-2s-1 and 70% relative humidity
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted and purified from leaves of at least 30 seedlings per plate using QIAGEN-RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to guidelines specified by the manufacturer. Two biological replicates were prepared for each combination. RNA was quantified using a Nanodrop-ND 8000 spectrophotometer (Thermo Fisher Scientific) and RNA quality was assessed by agarose gel analysis and using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. Samples with the RNA integrity number of 7.5 and above were chosen for further analysis.
|
| Label |
cy3
|
| Label protocol |
Microarray analysis was performed using Agilent-021169 Arabidopsis 4 Oligo Microarray (V4) (Probe Name version). In total 150ng of total RNA was used to prepare Cyanine-3 (Cy3) labeled probe with the help of low RNA input linear amplification/labeling kit (Agilent technologies) and 1.65µg of Labeled cRNA probes were fragmented using fragmentation buffer (Agilent Technologies) and hybridized to the Arabidopsis arrays according to manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
Microarray analysis was performed using Agilent-021169 Arabidopsis 4 Oligo Microarray (V4) (Probe Name version). In total 150ng of total RNA was used to prepare Cyanine-3 (Cy3) labeled probe with the help of low RNA input linear amplification/labeling kit (Agilent technologies) and 1.65µg of Labeled cRNA probes were fragmented using fragmentation buffer (Agilent Technologies) and hybridized to the Arabidopsis arrays according to manufacturer’s instructions.
|
| Scan protocol |
The arrays were scanned using the high resolution array scanner (Agilent technologies) with the appropriate settings for the one color gene expression arrays after hybridization.
|
| Description |
Gene expression at 4 degree for 3 h
|
| Data processing |
Array images were acquired with Agilent's dual-laser microarray scanner and signal intensities were extracted from the scanned images with dedicated Agilent Feature Extraction software (Agilent technologies). The outliers and the abnormal features were flagged and the data was normalized using the intra-array percentile shift normalization (threshold of 75 and above) and median based inter-array normalization. The GeneSpring software (Agilent technologies) was used to calculate the intensity ratios and fold changes.
All the genes with a P value below 0.05 and a fold change above 2 were chosen for further analysis.
|
| |
|
| Submission date |
Nov 12, 2014 |
| Last update date |
Jun 30, 2016 |
| Contact name |
Zhulong Chan |
| E-mail(s) |
zhulongch@wbgcas.cn
|
| Organization name |
Chinese Academy of Sciences
|
| Department |
Wuhan Botanic Garden
|
| Street address |
Moshan, Wuchang District
|
| City |
Wuhan, Hubei Province |
| ZIP/Postal code |
430074 |
| Country |
China |
| |
|
| Platform ID |
GPL9020 |
| Series (2) |
| GSE63184 |
RNA-DIRECTED DNA METHYLATION 4 modulates cold stress resistance in Arabidopsis through the C-REPEAT-BINDING FACTOR-mediated pathway [Agilent] |
| GSE63186 |
RNA-DIRECTED DNA METHYLATION 4 modulates cold stress resistance in Arabidopsis through the C-REPEAT-BINDING FACTOR-mediated pathway |
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