|
| Status |
Public on Feb 28, 2017 |
| Title |
B5_CRY_3B7N_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
chemical treatment: 3-bromo-7-nitroindazole
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
developmental stage: seedlings
genotype: cry1cry2
|
| Treatment protocol |
Seeds sown on medium with respective chemical treatment at 15 µM were kept at 4°C in dark for 3 days followed by continuous blue light irrradiation for 4 days at intensity of 5 µmol m-2s-1.
|
| Growth protocol |
Seeds of Arabidopsis thaliana were sterilized with sodium hypochlorite solution and EtOH and washed with sterilized water. Sterilized seed were sown on medium comprised of 0.5x MS salt mixture, MES, 1x Gamborg’s vitamin mixture, respective chemical (added below 50°C) and 0.8 % (w/v) agar (pH 5.8) and incubated in the dark at 4˚C for 3 days. Subsequently, the seeds were incubated under continuous blue light for 4 days. 4-day-old seedlings were harvested and frozen in liquid nitrogen immediately.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted using RNeasy Plant Mini RNA kit (Qiagen, USA) according to the manufacturer’s instructions.in brief, 0.2 g of frozen tissues was grinded using mortar and pestle and bind and total RNA was allowed to bind to RNeasy Mini Spin columns and further followed by washing by buffer and finally eluted using RNase-free water. Eluted RNAs was then treated with of RNase-free DNase I (Takara, Japan) and extracted with phenol:chloroform.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 1 ug RNA using Agilent One-Color Microarray Quick-Amp Labeling kit according to the manufacturer's instructions. Cy3-labeled cRNA was purification using Qiagen RNAeasy column purification. Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 μg of purified Cy3-labelled cRNA were fragmented in reaction mixture containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent at 60°C for 30 min in a reaction volume of 55 mL. After incubation, the reaction was immediately stopped with addition of 55 mL 2X GEx Hybridization Buffer HI-RPM. Preapared cRNAs were lay on slides and microarray chip was incubated in oven at 65°C for 17 hours with rotation. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately scanned after washing using the Agilent DNA Microarray Scanner (G2505B) with one color scan setting for 1x44k array slides
|
| Description |
description
Gene expression of 4d blue light irradiated cry1cry2 seedlings in 3-bromo-7-nitroindazole treatment
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Oct 18, 2016 |
| Last update date |
Feb 28, 2017 |
| Contact name |
Wen Dee Ong |
| E-mail(s) |
wendee.ong@riken.jp
|
| Organization name |
RIKEN
|
| Department |
Biomass Engineering
|
| Lab |
Synthetic Genomic Research Group
|
| Street address |
1-7-22 Suehiro-cho
|
| City |
Yokohama |
| State/province |
Kanagawa |
| ZIP/Postal code |
230-0045 |
| Country |
Japan |
| |
|
| Platform ID |
GPL9020 |
| Series (2) |
| GSE88864 |
Genes expression analysis of chemical-induced hypooctyl elongation under blue light irradiation |
| GSE88889 |
Chemical-induced inhibition of blue light-mediated seedling development in Arabidopsis |
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