Individual leaves were infiltrated in the morning using a needle-less syringe with 1x10E8 Pseudomonas syringae pv. tomato DC3000 hrcC and harvested 9h later.
Growth protocol
Plants were grown in pots with BM-2 soil (Berger Peat Moss Ltd, Quebec, Canada) at a density of 9 plants per pot and kept at 22 degrees Celsius and75% humidity with a 12 hour day length.
Extracted molecule
total RNA
Extraction protocol
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
Label
Alexa555
Label protocol
Following procedure was performed with Amino Allyl MessageAmp aRNA Amplification Kit II (Ambion).First, cDNA was synthesized from 1ug of total RNA. Next, RNA was in vitro transcribed. The RNA was then labeled with Alexa555 in coupling reaction.
Hybridization protocol
Labeled RNA was suspended in hybridization buffer (50% Formamide, 5xSSC, 0.1% SDS, 10 ug Sheared Salmon Sperm DNA (Eppendorf), 50pg calbation oligo)
Scan protocol
Slide was scaned using Genepix4000B with low and high voltages of photomultiplier at 532nm and constant voltage at 635nm.
Description
cbp60g,cbp60h,pad4 triple infiltrated with 1x10E8 Pseudomonas syringae pv. tomato DC3000 hrcC, harvested 9h after infiltration in setset2 experiment.
Data processing
The median of ratios for each spot was processed by the following linear model: Sij = mu + Ai + Bj + Eij where Sij denotes the log2-transformed, median of ratios for the spot, mu denotes a constant, Ai and Bj denote the effects of i-th gene and j-th subarray. The residual Eij is assumed to be independent and normally distributed. Stable genes-based quantile normalization was applied for slide-to-slide comparisons. set1_col_hrcC_9h, set1_col_psm_24h, set1_double_Psm_24h, set1_double_hrcC_9h, set1_pad4_Psm_24h, set1_pad4_hrcC_9h, set1_triple_Psm_24h, set1_triple_hrcC_9h, set2_col_hrcC_9h, set2_col_psm_24h, set2_double_hrcC_9h, set2_double_psm_24h, set2_pad4_Psm_24h, set2_pad4_hrcC_9h, set2_triple_Psm_24h, set2_triple_hrcC_9h, set3_col_Psm_24h, set3_col_hrcC_24h, set3_double_Psm_24h, set3_double_hrcC_24h, set3_pad4_Psm_24h, set3_pad4_hrcC_24h, set3_triple_Psm_24h, set3_triple_hrcC_24h, set1_052422_hrcC_9h, set2_052422_hrcC_9h, and set3_052422_hrcC_9h were normalized together. Perl scripts for the linear model and the normalization are available for non-commercial research conducted upon request (Fumiaki Katagiri, katagiri@umn.edu). Spots with bad quality (missing, lints, and high background) were flagged as indicated in Flag in the attached .gpr file for this sample.
