|
| Status |
Public on Dec 19, 2007 |
| Title |
Whole roots 2hr KCl control treated then incubated in protoplast-generating solution minus enzymes, biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
12 day old Arabidopsis roots, 2hr KCl control treated then incubated in protoplast-generating solution minus enzymes
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
Genotype: Col-0
All root cells
|
| Biomaterial provider |
Col-0 (Lehle Seeds)
|
| Treatment protocol |
At dawn on 12th day plants were either transitory/continuous nitrogen-treated (KNO3 to 5mM added) or KCl treated (KCl to 5mM added) as a control treatment for 2 hrs. To isolate single cell types (LRC, EpiC, EndoP, Peri, Stele) cells were incubated in a solution containing cell-wall digesting enzymes and then Fluorescence Activated Cell Sorting (FACS) sorted as Birnbaum et al (2005) Nature Methods 2: 615-619. To isolate mock FACS-sorted protoplasts the same proceedure was followed but the cells were not FACS-sorted, instead the protoplasts were incubated on ice for the time it takes to FACS-sort (1 hour). After FACS or mock-FACS cell sorting cells were frozen using liquid nitrogen. 5mM KNO3 was either maintained in the protoplast-generating solution (continuous treat) or not (transitory treat) to test how to best preserve the cellular nitrogen-response during the 1 hr protoplast generating procedure. As well as the 'protoplasts' controls we harvested whole roots that were either frozen using liquid nitrogen directly after the 2hr treatment or 3.5hr treatment, or whole roots incubated in protoplast-generating solultion (minus enzymes; transitory or continuous treated) and then frozen. 1mM MSX in combination with 5mM KNO3 (or with 5mM KCl as a control), or also with 5mM glutamine (Gln) for 2 hrs was used to block and then release the block of the assimilation of KNO3 in order to seperate KNO3 effects from assimilated-nitrogen effects in FACS-sorted pericycle cells.
|
| Growth protocol |
Plants were grown for 12 days in long day (16hr light/8hr dark) 22 oC conditions in a Percival (Percival Scientific). Plants were grown hydroponically in basal MS containing no nitrogen except 0.5mM ammonium succinate and 3mM sucrose. All plant lines are in the Col-0 background.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was either extracted using the Plant RNAeasy kit (Qiagen, Hilden, Germany) for cell samples (LRC, EpiC, EndoP, Peri, Stele, Protoplasts), or for whole root samples using TRIzol reagent (Invitrogen, Carlsbad, CA). Protocols were followed according to manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA from all isolated cell samples (LRC, EpiC, EndoP, Peri, Stele) were prepared according to the standard two-cycle Affymetrix protocol from 50 ng total RNA, or from all whole root samples and protoplast samples according to the standard one-cycle Affymetrix protocol from 1 ug total RNA.
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| |
|
| Hybridization protocol |
Following fragmentation, 8 ug of cRNA were hybridized for 16 hr at 45C on an Affymetrix ATH1 GeneChip Arabidopsis Whole Genome Array in the Affymetrix Hybridization oven 640 following standard Affymetrix protocol. GeneChips were washed and stained using the 'EukGE-WS2v4_450' protocol in the Affymetrix GeneChip Fluidics Station 450.
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| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip 3000 Scanner according to standard Affymetrix protocols.
|
| Description |
n/a
|
| Data processing |
The data were analyzed with dChip using dChip default analysis settings and 'Average' as the normalization model, PM match only. Note that samples were normalized in two batches according to the sample type: Cell type specific samples (columns B-BC), and protoplasts and whole roots (columns BD-CG), all using the procedure described above. Non nuclear-encoded or ambiguous probe-gene matches were not used (according to latest annotation file from Affymetrix (April 2007). If the signal was log2 less than 6 in both treat and control experiments probes were removed from analysis; the value of 6 based on expression values of previously documented cell-specific genes. Probes determined to be affected by protoplast generation procedure were not analysed.
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| |
|
| Submission date |
Apr 26, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Kenneth David Birnbaum |
| E-mail(s) |
ken.birnbaum@nyu.edu
|
| Phone |
212-998-8257
|
| Organization name |
New York University
|
| Department |
Biology
|
| Lab |
Birnbaum
|
| Street address |
12 Waverly Place
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10003 |
| Country |
USA |
| |
|
| Platform ID |
GPL198 |
| Series (1) |
| GSE7631 |
Cell-specific nitrogen responses in the Arabidopsis root |
|
| Relations |
| Reanalyzed by |
GSE119083 |
