|
| Status |
Public on Jun 01, 2011 |
| Title |
Tomato fruit wound inoculation by Colletotrichum coccodes 2 |
| Sample type |
RNA |
| |
|
| Source name |
Tomato breaker fruit parenchyma cells
|
| Organism |
Solanum lycopersicum |
| Characteristics |
cultivar: Hazera 1402
tissue: pericarp
|
| Treatment protocol |
The fruits had been surface-sterilized in 0.3% (v/v) hypochlorite for 10 min, rinsed thoroughly with water and dried. Fruit inoculation was carried out on the pericarp. A 0.5mm thick, 10-mm diameter of the peel tissue was removed from the tomato fruit and replaced by a 0.5mm thick hyphal disk of Colletotrichum coccodesmycelia (Alkan et al., 2008). This hyphal disk was obtained by growing spores for 3 days on primary media and transferred to 1 day growth on secondary media (“hyphal mat infection method”) (Alkan et al., 2008).
|
| Growth protocol |
The plants were grown in pots filled with a peat-vermiculite (4:1, v/v) mixture containing slow-release high-N Multicote 4 with microelements (0.3% w/w; Haifa Chemicals, Haifa Bay, Israel). Average temperatures in the greenhouse during the growth period fluctuated from 18 to 25˚C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from the fruit pericarp, of 10 mm diameter and 3-4 mm deep. Each biological repeat consisted of 20 infection or treatments front pericarp of 10 mm diameter and 3-4 mm deep from at least five different fruit. For RNA isolation one grams of tissue was ground in liquid nitrogen, transferred to 4ml extraction buffer (100mM Tris-HCl at pH 9.0, 200mM NaCl, 15mM EDTA at pH 8.0, 0.5% sarkosyl) and homogenized, 4ml phenol was added flowed by 280µl of 3M sodium-acetate and homogenized. The extraction was subjected to 10min centrifuge at 10000rpm and 4ml of tri-reagent was added to the supernatant and flowed by another centrifuge. 4ml of isopropanol was added to the supernatant and kept in ice for an hour flowed by another centrifuge. The resulted pellet was resuspended with 1ml DEPC water, 0.5ml of 8N Licl was added and kept on ice for overnight. The resuspended pellet was centrifuge, treated with 70% ethanol and dried. The RNA pellet was treated with DNase (Turbo-DNA Free; Ambion, Austin, Texas). Followed by RNA cleanup with RNAeasy Plant Mini Kit (Qiagen, Hilden, Germany).
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 10 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix), in the Weizmann Institute microarray unit.
|
| |
|
| Hybridization protocol |
Following fragmentation, 12 ug of cRNA were hybridized for 16 hr at 45 degrees C on Affymetrix Tomato GeneChip. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using Affymetrix GC3000 7G.
|
| Description |
Tomato breaker fruit parenchyma cells wound inoculation by C. coccodes
|
| Data processing |
RMA via PARTEK software.
|
| |
|
| Submission date |
May 26, 2010 |
| Last update date |
Jun 01, 2011 |
| Contact name |
Robert Fluhr |
| E-mail(s) |
robert.fluhr@weizmann.ac.il
|
| Phone |
972-8-9342175
|
| Organization name |
Weizmann Institute of Science
|
| Department |
Plant Sciences
|
| Street address |
|
| City |
Rehovot |
| ZIP/Postal code |
76100 |
| Country |
Israel |
| |
|
| Platform ID |
GPL4741 |
| Series (1) |
| GSE21999 |
Tomato fruit wound inoculation by C. coccodes and ammonium treated |
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