strain: Wild type s time: 1h growth condition: harvested after 44h at 32°C and transfer to 26°C for 1 hour, M2 media covered with a sheet of cellophane mating type: mat+
Growth protocol
P. anserina strains used for the common reference are the wild type S strains of either mat+ or mat- mating type. P. anserina strains used for the experiment are the wild type s mat+ and the het-R het-V self-incompatible mat+ strains. The het-R het-V self-incompatible strain was obtained from the progeny of a cross between the het-r het-V and the het-R het-V1 parental strains. The het-R het-V strain grows normally at 32°C and undergoes cell death at 26°C. For RNA extraction, wild type and self-incompatible strains were grown on solid M2 medium overlaid with a cellophane sheet at 32°C for 44h. The growth medium was renewed 4h before the end of the culture at 32°C (transfer of the cellophane sheet on fresh prewarmed (32°C) M2 medium). After growth at 32°C the strains were either collected (time T0) or transferred to 26°C on prewarmed M2 medium for 30min (T30min), 1hour (T1h), 2 hours (T2h), 3 hours (T3h) and 4 hours (T4h). M2 is a minimal synthetic medium as described (Esser K.,1974; pp. 531-51 in Handbook of Genetics).
Extracted molecule
total RNA
Extraction protocol
Strains were grown on cellophane sheet (cat#1650193, BioRad, Marne la Coquette, France) or cheesecloth (Sefar Nitex 03-48/31, Dominique Dutscher, Brumath, France) placed on Petri-dishes. In order to collect sufficient amounts of biological materials (20 to 100 mg), 1 to 5 Petri-dishes were simultaneously inoculated. Biological material was scraped from cellophane sheets or cheesecloth and flash frozen with buffer RLT (Qiagen, Courtaboeuf, France) in liquid nitrogen. The biological material was grinded with a Mikro-Dismembrator (Sartorius, Aubagne, France) in nitrogen freezed vessels. After removing cell debris by centrifugation, nucleic acids were passed through a Qiashreddrer (Qiagen) for sharing DNA and total RNA was purified on RNeasy Plant Mini Kit columns (Qiagen), with an additional DNase treatment. The quality and quantity of the total RNA was determined by using a NanoDrop ND-1000 spectrophotometer and the Bionalyzer 2100 system (Agilent).
Label
Cy3
Label protocol
Labelling experiments were done following the two-color microarray-based gene expression analysis instructions (version 5.0, February 2007) as described by the manufacturer (Agilent). One microgram aliquots of total RNA were amplified and Cy-labelled with the Agilent’s Low RNA input fluorescent linear amplification (LRILAK) PLUS kit (Agilent) along with Agilent's Two-Color RNA Spike-in Kit. The labelling efficiency and the product integrity were checked by using a NanoDrop ND-1000 spectrophotometer and the Bionalyzer 2100 system (Agilent)
Channel 2
Source name
Common reference (a pool of four biosources: M48h, M96h, C48h and C96h)
biosource 1: mat+ and mat- S strain harvested after 48 hours of growth on M2 media covered with a sheet of cellophane. name biosource 1: M48h biosource 2: mat+ and mat- S strain harvested after 96 hours of growth on M2 media covered with a sheet of cellophane. name biosource 2: M96h biosource 3: mat+ and mat- S strain cultivated for 96 hours on M2 media covered with a cheesecloth, spermatization with mat- and mat+ (respectively) S spermatia and harvesting after 48 h. name biosource 3: C48h biosource 4: mat+ and mat- S strain cultivated for 96 hours on M2 media covered with a cheesecloth, spermatization with mat- and mat+ (respectively) S spermatia and harvesting after 96 h. name biosource 4: C96h
Growth protocol
P. anserina strains used for the common reference are the wild type S strains of either mat+ or mat- mating type. P. anserina strains used for the experiment are the wild type s mat+ and the het-R het-V self-incompatible mat+ strains. The het-R het-V self-incompatible strain was obtained from the progeny of a cross between the het-r het-V and the het-R het-V1 parental strains. The het-R het-V strain grows normally at 32°C and undergoes cell death at 26°C. For RNA extraction, wild type and self-incompatible strains were grown on solid M2 medium overlaid with a cellophane sheet at 32°C for 44h. The growth medium was renewed 4h before the end of the culture at 32°C (transfer of the cellophane sheet on fresh prewarmed (32°C) M2 medium). After growth at 32°C the strains were either collected (time T0) or transferred to 26°C on prewarmed M2 medium for 30min (T30min), 1hour (T1h), 2 hours (T2h), 3 hours (T3h) and 4 hours (T4h). M2 is a minimal synthetic medium as described (Esser K.,1974; pp. 531-51 in Handbook of Genetics).
Extracted molecule
total RNA
Extraction protocol
Strains were grown on cellophane sheet (cat#1650193, BioRad, Marne la Coquette, France) or cheesecloth (Sefar Nitex 03-48/31, Dominique Dutscher, Brumath, France) placed on Petri-dishes. In order to collect sufficient amounts of biological materials (20 to 100 mg), 1 to 5 Petri-dishes were simultaneously inoculated. Biological material was scraped from cellophane sheets or cheesecloth and flash frozen with buffer RLT (Qiagen, Courtaboeuf, France) in liquid nitrogen. The biological material was grinded with a Mikro-Dismembrator (Sartorius, Aubagne, France) in nitrogen freezed vessels. After removing cell debris by centrifugation, nucleic acids were passed through a Qiashreddrer (Qiagen) for sharing DNA and total RNA was purified on RNeasy Plant Mini Kit columns (Qiagen), with an additional DNase treatment. The quality and quantity of the total RNA was determined by using a NanoDrop ND-1000 spectrophotometer and the Bionalyzer 2100 system (Agilent).
Label
Cy5
Label protocol
Labelling experiments were done following the two-color microarray-based gene expression analysis instructions (version 5.0, February 2007) as described by the manufacturer (Agilent). One microgram aliquots of total RNA were amplified and Cy-labelled with the Agilent’s Low RNA input fluorescent linear amplification (LRILAK) PLUS kit (Agilent) along with Agilent's Two-Color RNA Spike-in Kit. The labelling efficiency and the product integrity were checked by using a NanoDrop ND-1000 spectrophotometer and the Bionalyzer 2100 system (Agilent)
Hybridization protocol
Hybridization experiments were done following the two-color microarray-based gene expression analysis instructions (version 5.0, February 2007) as described by the manufacturer (Agilent). 825 ng of each of the Cy3- and Cy5-labeled targets were mixed and incubated on an Agilent microarray slide for 17 hours at 65°C, in a rotating oven at 10 rpm for a 4x44K array format, using an Agilent in situ hybridization kit. The slides were washed in Agilent’s Gene Expression Wash Buffers following manufacturer’s instructions, then, any traces of solution were removed by centrifugation at 800 rpm for 1 min.
Scan protocol
Microarrays were scanned using the Agilent DNA microarray Scanner model G2567AA at 5 microns resolution using the extended dynamic range (XDR) feature.
Description
Biological replicate 3 of 4. Zone 1 of WT
Data processing
Spot and background intensities were extracted with the Feature Extraction (FE, v9.5.3) software (Agilent) using the GE2-v4_95_Feb07 default protocol. Preliminary array quality was assessed through the use of Agilent control features as well as spike-in controls (Agilent 2-Color Spike-in Kit for RNA experiment). Subsequent flagging was done according to the GenePix Pro software (Molecular Devices Syunnyvale, CA, USA) nomenclature, including four levels of flags (good (100), bad (-100), not found (-50), moderate (0)). FE software normalized data (Local background was subtracted and LOWESS normalization) from arrays issued of GPL10116 platform were processed with the MAnGO. Moderate t-test with adjustment of p-values was computed to measure the significance with each expression difference.