cultivar: Nipponbare tissue: roots growth period: 18 days genotype: Osgs1;1
Treatment protocol
Germinated seeds were hydroponically grown under one-quarter strength basal nutrient solution lacking a nitrogen (N) source with 1 mM of ammonium for 18 days.
Growth protocol
We grew samples using two different conditions. Growth condition 1 for SAMPLE 1-17: The conditions in the greenhouse at Tohoku University, Sendai, Japan were: 14 h of light at 26oC and 10 h of darkness at 23oC without humidity control; light strength, 810 μmol∙m-2∙sec-1; light source, metal halide lamp (05:30 - 18:30) and sunlight. WT-, heterozygous Osgs1;1-, and homozygous Osgs1;2 seeds were sterilized in 3 steps: (i) seeds were immersed in 60oC water for 10 min and then washed in flowing water for 10 min, (ii) immersed in 70% ethanol for 30 sec and washed immediately, and (iii) immersed in 2% hypochlorous acid for 20 min and then washed in water for 20 min. Then they were incubated at 30oC for 2 days in the dark. Germinated seeds were hydroponically grown for 18 days in one-quarter strength basal nutrient solution that lacked an N source and contained 150 mM NaH2PO4•2H2O, 75 mM K2SO4, 75 mM CaCl2•2H2O, 100 mM MgCl2•H2O, 11.25 mM Fe•EDTA, 12.5 mM H3BO3, 2.25 mM MnSO4•5H2O, 0.075 mM CuSO4•5H2O, 0.175 mM ZnSO4•7H2O, and 0.025 mM Na2MoO4•2H2O and 1.0 mM NH4Cl to harvest root samples. Growth condition: Plants were hydroponically grown for 18 days.
Extracted molecule
total RNA
Extraction protocol
Total RNA isolation was performed using RNeasy Plant Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instruction.
Label
biotin
Label protocol
Biotinylated cRNA was labeled using a biotinylated nucleotide analog/ribonucleotide mix using GeneChip 3’ IVT Express Kit (Affymetrix, Santa Clara, CA, USA).
Hybridization protocol
Following fragmentation, 12.5 ug of cRNA were hybridized for 16 h on GeneChip Rice Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450 (Affymetrix).
Scan protocol
GeneChips were scanned using the GeneChip scanner 3000 integrated with Affymetrix® Microarray Suitesoftware.
Description
The sample was grown and harvested at Tohoku university. For details, see metadata as described in Kusano et al. SAMPLE 4
Data processing
The microarray data normalization and background correction was performed using robust multi-array average (RMA) with Bioconductor package 'simpleaffy' by using R/Bioconductor.
