A genome wide microarray, covering the 10546 presently known and predicted CDS, has been constructed with the Agilent in-situ synthesized 60-mers technology.
1) A library of 10 oligonucleotides per CDS has been design by the manufacturer.
2) A computational step has been performed to determine 3' position, intron position and cross-hybridization rate of all probes. These data have been compiled to attribute a score to each probe and thus, to select a first set of 4 oligonucleotides per CDS.
3) Microarray v.2 (GPL10115), manufactured with this design, have been used for a selection procedure based on the use of transcriptomic experimental data generated with diverse culture conditions. To maximize the number of expressed CDS, experimental data were generated with a large range of growth conditions covering crucial physiological changes from key steps of vegetative growth and sexual development.For each condition, four biological replicates were done to increase the statistical power of the analysis. An oligonucleotide scoring has been settled to select the best oligonucleotide per CDS.
The final array design, Microarray v.3 (GPL10116), contained 10,539 probes for nuclear CDS. The array was enriched with 17 mitochondrial CDS probes that did not undergo the experimental screening. Each probe was replicated four times on each 44K array to improve the statistical significance of results. Before using in industrious transcriptomic experiment, ultimate qualification steps have been carried out. First, DNA extracted from the/ S/ and /s /strains was hybridized with the final design Microarray v.III. The apha_ORF and /het/ genes were identified as polymorphic between /s/ and /S/ strains (|FC|>2; a/ =/0.001) confirming previous genetic and molecular analyses (Turcq B etal.,1990*,* /Current genetics, /Sellem et al., 1990, Mol. Gen. Genet.). In second qualification step, a self-to-self hybridization experiment, replicated three times, has been /per/formed with a commonreference: 89.3% of the probes displayed a signal-to-standard-deviation ratio >3. The final microarray design is referenced as AMADID 018343. Microarray slides are available to the research community from Agilent.
Overall design: We developed oligonucleotide microarrays for the filamentous fungus Podospora anserina as part of the ANR program project grant ANR-05-BLAN-0385 (project SexDevMycol), coordinated by R. Debuchy. This project was aimed to analyze the sexual development of /P. anserina/ with a variety of approaches, including transcriptomic profiling. Different versions of microarrays were tested on mycelium and sexual development time courses, to evaluate their perfomance in comparing transcriptomic profiles of vegetative vs sexual stages, and in analyzing transcriptomic profiles evolution during sexual development. Complete analyses were performed with the microarray final version (v.III) and several manuscripts dealing with these topics are in preparation.
The s (small s) and S (big S) strains of Podospora anserina are different geographical isolates of the same species. Both strains can cross and have identical ITS (accession #: AY278557 and GU391422) confirming that they belong to the same species. The S strain has been completely sequenced (Espagne et al., Genome Biology, 2008, 9, R77 ) and its genome is currently at the finishing step (P. Silar and P. Wincker, unpublished results). The genome of the s strain is not sequenced, and only a few genes are known. The major known difference is at the level of the s locus controling vegetative incompatibility (Turcq et al., Current genetics 1990, 17:297-303).
PART1 (with GPL10115): reference design, 5 conditions each with four biological replicates : M24h, M48h, M96h, C24h, C48h; common reference is a pool of four conditions M48h, M96h, C48h and C96h ; Conditions are labelled in Cy3 and the common reference in Cy5
PART2 (with GPL10116): DNA hybridization with a direct design, wt s versus wt S, dye swap (four technical replicates); RNA Homotypic hybridization with the common reference sample (three technical replicates)
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