|
| Status |
Public on Jun 29, 2022 |
| Title |
rep3_200N_Col-0 |
| Sample type |
RNA |
| |
|
| Source name |
14d seedlings, Col-0, 200 mM NaCl plate, replicate 3
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: whole seedling
|
| Treatment protocol |
0N: seeds were sow in MS plates containing no NaCl. 200N: seeds were sow in MS plates containing 200 mM NaCl.
|
| Growth protocol |
all seeds were sterilized and subjected to cold pretreatment at 4℃ for 3 days in dark before sowing in MS plates, which contains the following compositions: half strength of MS (Murashige and Skoog 1962), B5 organic compound (Gamborg et al. 1968), 1% sucrose, 0.05% MES [2-(N-morpholino)ethanesulfonic acid monohydrate]. After adjusting the pH to 5.7, 7 g/L phytoagar (Duchefa Biochemie) was added. The plants were grown under 16h/8h day/night photoperiod with a light intensity of approximately 50 µmol m-2s-1. 14-day-old seedlings were harvested in liquid N2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the Qiagen RNeasy Plant Mini Kit (Qiagen, Hilden, DE) following the manufacturer's recommendations. This protocol includes an on-column DNase digestion using the RNAse-Free DNase Set (Qiagen, Hilden, DE) to digest contaminating DNA in RNA solutionsd. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
0.2 μg of total RNA was amplified by a Low Input Quick-Amp Labeling kit (Agilent Technologies, USA) and labeled with Cy3 (CyDye, Agilent Technologies, USA) during the in vitro transcription process. 1.65 μg of Cy3-labled cRNA was fragmented at 60°C for 30 minutes.
|
| |
|
| Hybridization protocol |
Correspondingly fragmented labeled cRNA is then pooled and hybridized to Agilent Arabidopsis V4 4*44K Microarray (Agilent Technologies, USA) at 65°C for 17 h.
|
| Scan protocol |
The microarrays are scanned with an Agilent microarray scanner (Agilent Technologies, USA) at 535 nm for Cy3. The array image was analyzed by the Feature Extraction software version 10.7.1.1 using the default setting.
|
| Description |
gene expression of 14d seedlings grown in 200 mM NaCl plates
|
| Data processing |
The CEL files were analyzed using GeneSpring (version 14.9.1, Agilent technology). The raw signal values employed a cutoff if the values in all samples were <100. Following the cutoff, Quantile normalization and baseline transformation to median of all samples were performed. The normalized genes were then statistically analyzed using two-way ANOVA and the p values were computing with multiple testing corrections of Benjamini-Hochberg False Discovery Rate (p value cutoff of 0.05). Three biological replicates were performed in this experiment. Gene expression passed 2-way ANOVA (p<0.05) were applied to fold change (FC) comparison.
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| |
|
| Submission date |
Jan 17, 2022 |
| Last update date |
Jun 29, 2022 |
| Contact name |
Wan-Hsing Cheng |
| E-mail(s) |
whcheng@gate.sinica.edu.tw
|
| Organization name |
Academia sinica
|
| Department |
IPMB
|
| Street address |
128 Academia Road, Section 2, Nankang
|
| City |
Taipei |
| ZIP/Postal code |
11529 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL9020 |
| Series (1) |
| GSE193841 |
Differential expression of genes involved in salt-treated iU1 seedlings |
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