Comment[ArrayExpressAccession] E-GEOD-54803 MAGE-TAB Version 1.1 Public Release Date 2014-02-12 Investigation Title Gene expression profiles in the bacterial pustule-resistant soybean cultivars(2) Comment[Submitted Name] Gene expression profiles in the bacterial pustule-resistant soybean cultivars(2) Experiment Description To investigate the differential action between resistance and susceptible cultivars, we examined genome wide expression levels at five time points after Xag-inoculation using microarray. The soybean microarray was designed using the server-based eArray platform (http://earray.chem.agilent.com/earray/) from Agilent Technologies (Wolber et al., 2006). The current slide layout consists of four arrays of >44,000 features, including a set of Agilent's positive and negative control features. An oligonucleotide microarray containing 42,564 unique probes (60-mer) was created from 33,574 unigenes from Glycine max database (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=3847) (May 2009) as templates for probe design. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Kim Kim Kim Choi Person First Name Jung Min Jung Seong-yun In-soo Person Mid Initials M Person Email jmkim2010@korea.com Person Affiliation NAR Center, Inc. Person Phone 82-10-3459-4776 Person Address NAR Center, Inc., 22-5 Daeheung-dong, Jung-gu, Daejeon, South Korea Person Roles submitter Protocol Name P-GSE54803-1 P-GSE54803-5 P-GSE54803-6 P-GSE54803-2 P-GSE54803-3 P-GSE54803-4 P-GSE54803-7 Protocol Description The average fluorescence intensity for each spot was calculated and local background was subtracted. All data normalization and selection of fold-changed genes were performed using GeneSpring GX 7.3 (Agilent Technology). Genes were filtered with removing flag-out genes in each experiment. Intensity-dependent normalization (LOWESS) was performed, where the ratio was reduced to the residual of the Lowess fit of the intensity vs. ratio curve. ID_REF = VALUE = log2 ratio test/susceptible PRE_VALUE = ratio test/susceptible For non-inoculated (Cy3) and inoculated (resistant or susceptible) (Cy5) soybean RNAs, the synthesis of target cRNA and hybridization were performed using AgilentM-bM-^@M-^Ys Low RNA Input Linear Amplification Kit PLUS (Agilent Technology) according to the manufacturerM-bM-^@M-^Ys instructions. After checking labeling efficiency, each 750 ng of cyanine 3-labeled and cyanine 5-labeled cRNA targets were mixed and the fragmentation of cRNA was performed by adding 10x blocking agent and 25x fragmentation buffer and incubating at 60 M-0C for 30 min. The fragmented cRNA was resuspended with 2x hybridization buffer and directly pipetted onto soybean microarray placed in Hybridization Chamber (Agilent Technologies). The arrays hybridized at 65 M-0C for 16 h using Agilent Hybridization Oven. The hybridized microarrays were washed as the manufacturerM-bM-^@M-^Ys washing protocol (Agilent Technology). In the green house experiment, individual plant was inoculated with the strain 8ra Xag. The inoculum was prepared by culturing in PDA (potato dextrose agar) medium. Bacterial cells on the culture plates were sterile distilled water and adjusted to optical density 0.3~0.5 (108 colony forming units (cfu) per milliliter) at 600 nm in UV/Visible spectrophotometer. Inoculations were conducted on recently expanded second trifoliate leaves of 20- to 25-day-old plants. Surface of the leaves were sprayed with inoculum suspensions until water-soaking appearance. For better development of disease symptoms after inoculation, green house condition was held at 28 M-0C and 100% relative humidity by spraying water and by covering another polypropylene vinyl to protect infected soybean plants from direct sunlight. We developed two sets of near-isogenic line (NIL) by selecting heterozygous F5 plants among pedigree breeding lines from the cross Saturn x SS97225 (Lee x Seunheuk). F5 pedigree lines were grown in 2006 and inoculated with Xag strain 8ra. Seed from F5 plants that were heterozygous for the disease resistance was harvested and F5:6 pedigree lines were grown in 2007. About 40 F6 plants from each segregating F5:6 lines were inoculated with the strain 8ra. F6 lines that were homozygous for the disease resistance were selected and harvested as F6:7 pedigree lines. About 40 F7 plant from each F6:7 lines were inoculated with the strain 8ra and homozygous for the disease resistance. Thus a NIL set consisted of F5 derived families homozygous phenotype for the disease resistance. Ten leaves, non-inoculated (control sample) and inoculated resistant or susceptible leaves at two time points, 3-hour, 12-hour, and 3-day (test groups, 2-condition x 3-time), for total RNA isolation were homogenized using liquid nitrogen with a mortar and pestle. Total RNA and genomic DNA from homogenized sample were extracted using TRIZOL reagent (GibcoBRL, Rockville, MD, USA) according to the protocol of the manufacturer. Total RNA was treated with the RNase-free DNase I (Promega, Madison, WI, USA) to reduce DNA contamination. Total RNA concentration and purity were determined spectrophotometrically by the absorbance ratio at 260:280 nm 1.8 or more. The integrity of total RNA samples was also ascertained by appearance of distinct 28S and 18S bands of ribosomal RNA using Bioanalyzer 2100 system (Agilent Technology, Santa Clara, CA, USA). The hybridization images were analyzed by Agilent DNA microarray scanner and the data quantification was performed using Agilent Feature Extraction software. Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name AGE ORGANISM PART Experimental Factor Type age organism part Comment[SecondaryAccession] GSE54803 Comment[GEOReleaseDate] 2014-02-12 Comment[ArrayExpressSubmissionDate] 2014-02-07 Comment[GEOLastUpdateDate] 2014-02-12 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-54803.sdrf.txt