|
| Status |
Public on Feb 28, 2013 |
| Title |
Guard cell protoplast_WT_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Guard cell protoplast, WT, replicate 3
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Columbia-0
tissue: Guard cell protoplast
|
| Treatment protocol |
Detached leaves were blended for 2 min and filtered through a 100 μm nylon mesh. Harvested epidermal peels were transferred to digestion solution 1 (0.5% (w/v) cellulase R-10 (Yakult Pharmaceutical Industry Co., Tokyo, Japan), 0.002% (w/v) pectolyase Y-23 (Seishin Pharmaceutical, Tokyo, Japan), 0.1% (w/v) polyvinylpyrrolidone K-30, 0.2% (w/v) bovine serum albumin (BSA), 0.25 M mannitol, 0.5 mM CaCl2, 0.5 mM MgCl2 and 10 mM MES-KOH, pH 5.5) and incubated at 25 °C for 3 h in a vigorously shaking water bath. After checking that mesophyll cell layers had been dissociated, the peels were transferred to digestion solution 2 (1.3% (w/v) cellulase RS, 0.0075% (w/v) pectolyase Y-23, 0.2% (w/v) BSA, 0.4 M mannitol, 0.5 mM CaCl2 and 0.5 mM MgCl2, pH 5.5) and incubated at 19 °C for 2 h. Protoplasts were filtered through four layers of 10 μm nylon mesh and collected by centrifugation at 1000 x g for 10 min. To remove mesophyll cell fragments and other contaminants, the protoplasts were subjected to density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, MO)
|
| Growth protocol |
Arabidopsis plants were grown on Jiffy-7 peat pellets (Jiffy Products International AS) under continuous light in a growth chamber at 23°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
QIAGEN Rneasy Plant Mini Kit used for total RNA isolation
|
| Label |
Cy3
|
| Label protocol |
Total RNA (200 ng) was used to prepare Cyanine-3 (Cy3) labelled probes using a low RNA input linear amplification/labeling kit (Agilent)
|
| |
|
| Hybridization protocol |
Labelled cRNA probes (1.65 µg) were fragmented using fragmentation buffer (Agilent) and hybridised to the whole Arabidopsis Gene Expression Microarray (V4, design ID 21169; Agilent Technologies) for 17 h at 65°C in a hybridization oven.
|
| Scan protocol |
The arrays were air-dried and scanned using the high-resolution array scanner (Agilent) with the appropriate settings for one-color gene expression arrays.
|
| Description |
Gene expression data from guard cell protoplasts of Col-0
|
| Data processing |
Raw data files were imported into GeneSpring 12 software (Agilent) and normalised using the intra-array percentile shift normalisation (threshold of 75 and above).
|
| |
|
| Submission date |
Jan 31, 2013 |
| Last update date |
Feb 28, 2013 |
| Contact name |
Juntaro Negi |
| E-mail(s) |
[email protected]
|
| Phone |
+81 92 642 2621
|
| Fax |
+81 92 642 2621
|
| URL |
http://plant.biology.kyushu-u.ac.jp/en/index.html
|
| Organization name |
Kyushu University
|
| Department |
Biology
|
| Street address |
6-10-1 Hakozaki, Higashi-ku
|
| City |
Fukuoka |
| ZIP/Postal code |
812-8581 |
| Country |
Japan |
| |
|
| Platform ID |
GPL12621 |
| Series (1) |
| GSE43964 |
Microarray analysis of scap1 mutant and wild type in Arabidopsis thaliana guard cell protoplast |
|
