|
| Status |
Public on Dec 06, 2016 |
| Title |
control, biological rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
QC cells sorted by pWOX5-GFP
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype/variation: plt1plt2 double mutant
tissue type: Quiescent Center (QC) cells
treatment: mock
treatment time: 1 hour
|
| Treatment protocol |
After treatment root tips were cut and harvested followed by protoplasting as described in Birnbaum, K. et al. Cell type-specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines. Nature methods 2, 615-619. Briefly: root tips were placed inside cell strainers (70-μm) in petri dishes containing protoplasting solution (50 ml of solution A (21.86 g mannitol, 150 mg KCl, 80 mg MgCl2, 59 mg CaCl2, 0.2 g BSA, 78 mg MES in 200 ml water, set to pH 5.5 with 1 M Tris), 750 mg cellulase, and 50 mg macerozyme) and incubated for 1 hour at room temperature with agitation. Protoplasts were collected by spinning down at 200x gravity. The cells were subsequently washed and filtered through 70-μm and 40-μm cell strainers with 350 µl solution A. Protoplasts expressing GFP were isolated in a fluorescence-activated cell sorter (FACSAria II, BD Biosciences). The nozzle size was 100 μm. Cells were sorted in phosphate-buffered saline (PBS, pH 7.4) as sheath fluid at an event rate of ~5000-10000 events per second and fluid pressure of 20 psi. Upon excitation at 488 nm, GFP expressing protoplasts were selected based on fluorescence emission in the green channel (530 nm with band width of 30 nm) compared to negative controls. Cells were sorted into the RLT lysis buffer (Qiagen) supplemented with β-mercaptoethanol (1%), mixed and flash frozen instantly.
|
| Growth protocol |
To determine the PLT2 regulated transcriptome in the QC we used the plt1;plt2 line harboring the dexamethasone inducible complementing PLT2::PLT2-GR construct and introduced a WOX5::erGFP construct to allow QC cell sorting. Seeds were plated densely in 3 rows on square petri dishes on 0.5x GM agar medium covered with nylon mesh (Sefar Nitex 03-15/10). At 5 days after germination, seedlings were transferred to new 0.5x GM agar plates containing 10 µM dexamethosone (dex) in DMSO and incubated for 1 or 4 hours. The control samples were transferred to new plates containing DMSO only and incubated for 1 hour.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from the sorted protoplasts with the RNeasy Micro kit (Qiagen).
|
| Label |
biotin
|
| Label protocol |
Outsourced at ServiceXS (Leiden, the Netherlands)
|
| |
|
| Hybridization protocol |
Outsourced at ServiceXS (Leiden, the Netherlands)
|
| Scan protocol |
Outsourced at ServiceXS (Leiden, the Netherlands)
|
| Description |
Gene expression data from control
|
| Data processing |
Analysis with R/Bioconductor. Arrays were RMA normalized and differential gene expression analysis was performed with the limma package version 3.24.15
|
| |
|
| Submission date |
Mar 31, 2016 |
| Last update date |
Dec 06, 2016 |
| Contact name |
Luca Santuari |
| Organization name |
Wageningen University
|
| Department |
Plant Sciences Group
|
| Lab |
Plant Developmental Biology
|
| Street address |
Droevendaalsesteeg 1
|
| City |
Wageningen |
| ZIP/Postal code |
6708PB |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL21674 |
| Series (2) |
| GSE79751 |
Induction of pPLT2-PLT2-GR in Quiescent Center (QC) cells |
| GSE79755 |
The PLETHORA gene regulatory network guides growth and cell differentiation in Arabidopsis roots |
|
