|
| Status |
Public on Dec 08, 2020 |
| Title |
Arabidopsis pistil_fresh_stage12_rep 3 |
| Sample type |
RNA |
| |
|
| Source name |
Arabidopsis pistils,removed stigma, stage 12,replicate 3
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
developmental stage: stage 12
tissuse: Pistil removed stigma
|
| Treatment protocol |
The stigma was removed, and collected under a stereoscope microscope. More than 3 μg pistils was collected at stage 9-10 (S1), stage 11 (S2), and stage 12 (S3) with three biological replicates
|
| Growth protocol |
Plants were grown in a 10:10:1 mix of peat substrate: vermiculite: perlite under a 16-h-light/8-h-dark photoperiod at 22 ± 2 °C. For growth on plates, surface-sterilized Arabidopsis seeds were placed on half-strength Murashige and Skoog (MS) basal medium. Plates were kept at 4 °C for 3 days, then transferred to a growth chamber (Percival, Boone, IA, USA) with a 16-h-light/8-h-dark photoperiod at 22 °C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the RNasey Mini Kit (Qiagen p/n 74104) following the manufacturer's recommendations.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 |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes following the manufacturers instructions. On completion of the fragmentation reaction, 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to the Agilent Arabidopsis (V4) Gene Expression Microarray (4*44K, Design ID:021169) for 17 hours at 65°C in a rotating Agilent hybridization oven. 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 after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Dec 07, 2020 |
| Last update date |
Dec 08, 2020 |
| Contact name |
Shi-Xia Yu |
| E-mail(s) |
shixiayu@sjtu.edu.cn
|
| Organization name |
Shanghai Jiaotong University
|
| Street address |
Dongchuan Road, Shanghai Minhang District No. 800
|
| City |
Shanghai |
| ZIP/Postal code |
200240 |
| Country |
China |
| |
|
| Platform ID |
GPL12621 |
| Series (1) |
| GSE162759 |
Gene expression during Arabidopsis ovule development at stages 9-12 |
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