ecotype: Columbia tissue: young leaves treatment: moderate drought treatment time: 1 day
Treatment protocol
For drought treatment, pellets were weighed before sowing to determine the amount of water in pellets at the beginning of the experiment. Controlled moderate drought (mDr) was maintained by giving plants water to keep the soil moisture level at 30% of field capacity, which is 200% or 2 g H2O g-1 dry soil. Water was withheld at 30 days after sowing (DAS). For progressive drought (pDr) treatment, plants were grown in a growth room as described above, water was withheld at 35 DAS, and pellets were allowed to dry and monitored by weighing the pellets until the required pDr level was reached.
Growth protocol
Arabidopsis (Columbia) seeds were sown in moistened peat pellets, stratified at 4°C for 2 days, and then transferred to a growth room kept at 10 hours light (100 µmole m-2 s-1) and 22°C.
Extracted molecule
total RNA
Extraction protocol
For RNA isolation, two biological replicate samples of 5 pooled plants were collected at early (day1) and late stage (day10) for mDr, and first day of wilting for pDr, along with controls for mDr and pDr. RNA was isolated using RNeasy Kit (Qiagen, USA). After that, genomic DNA was eliminated using DNase kit (Qiagen, USA).
Label
biotin
Label protocol
RNA preparation/labeling was done using the Affymetrix 3' IVT Express kit, with biotin as label. The cRNA (or aRNA) is purified via beads and fragmented before hybridization.
Hybridization protocol
4 µg RNA samples were hybridized to the Affymetrix (ATH1 25K) GeneChip. The hybridization protocol was done using the GeneChip Hybridization, Wash, and Stain kit. The cocktail includes 12.5 ug aRNA, control oligo B2, 20x hyb controls of bioB, bioC, bioD, and cre, 2X hyb mix, and DMSO. The hybridization mix was heated at 99C for 5 min, 45C for 5 min, and then hybridized for 16h at 45C.
Scan protocol
Arrays were stained using the Affymetrix FS450 fluidics station with FS450_0001 fluidics protocol. Afterwards, arrays were scanned with the Affymetrix Gene Chip Scanner 3000 with HR (high resolution).
For each of the drought experiments, mDr-Day1, mDr-Day10 and pDr, raw data were background corrected, normalized and summarized according to the custom CDF using RMA (Irizarry et al., 2003). RMA was implemented in the 'affy' Bioconductor package within the R statistical computing environment.
Each of the mDr-Day01, mDr-Day10 and pDr sample groups (along with their controls, for a total of 4 samples per group) were normalized separately.
The custom CDF was built by uniquely mapping 232,697 probe sequences to 21,389 Arabidopsis (TAIR8) gene-based probe sets in the following manner: (i) probes that have perfect sequence identity with a single target gene were selected, (ii) probes mapping to reverse complements of genes were annotated separately as antisense probes (not used in the above counts), and finally, (iii) probes were grouped into probe sets, each corresponding to a single gene, and probe sets having at least 3 unique sequences were retained (>99% probe sets have >=5 probes). Note that these stringent criteria used to construct the CDF make it possible to reliably measure expression values of members of multigene families (free from cross-hybridization between paralogs showing high sequence similarity).
