Four-week-old Arabidopsis thaliana ecotypes Columbia (Col-0) were treated with either 150 mM NaCl or 10 μM ABA for 6 hours. The whole seedlings including root and shoot were harvested and pooled for each treatment before freezing in liquid nitrogen and storage at -80ºC for RNA isolation.
Growth protocol
Arabidopsis thaliana ecotypes Columbia (Col-0) seedlings grown in freshwater-irrigated pots filled with the artificial soil (Vermiculite: perlite: soil = 3:1:1, v/v) in a culture condition of 70% (±5%) relative humidity, 16 h 120 μmol•m-2•s-1 light / 8 h dark photoperiod at 22°C (±2°C).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIZOL Reagent (Cat#15596-018，Life technologies, Carlsbad, CA, US) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US). Qualified total RNA was further purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany).
Label
Cy3
Label protocol
Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Cat#5190-2305, Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany).
Hybridization protocol
Each Slide was hybridized with 1.65μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat#5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat#G2545A, Agilent technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit (Cat#5188-5327, Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions.
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat#G2565CA, Agilent technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution=5μm, PMT 100%, 10%, 16bit. Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US) Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
Description
Gene expression after 6h in NaCl-treatmented Arabidopsis seedlings
Data processing
The scanned images were analyzed with R-software (The R Project for Statistical Computing, http:// www. r-project.org/) of SAS system statistics to obtain background subtracted and spatially detrended Processed Signal intensities.
