2µg of mRNA for each time-point were used for cDNA synthesis by reverse transcription with incorporation of aminoallyl-dUTP (Sigma), then labeled by coupling to N-hydroxysuccinimidyl ester of Cy5 dye (Amerscham) and purified using QIAquick PCR purification kit.
Total RNA was isolated using a phenol extraction method. Frozen cells stored at -80°C were resuspended in 500ml TES buffer (20mM Tris HCl pH 8.5, 10mM EDTA, 1% SDS), 500ml acid phenol/chloroform/isoamylalcohol (25:24:1) and 200ml glass beads. The cells were vortexed vigorously for 30mn at 4°C. After centrifugation at 13000rpm at 4°C, aqueous phase was removed and 2 to 3 successive acid phenol/chloroform extraction were performed, followed by washing with 500ml chloroform/isoamyl alcohol (24:1). RNA was precipitated in 2 volumes of 95% ethanol and 1/5 volume 1M ammonium acetate, resuspended in 500ml DEPC-treated H2O. Second precipitation was performed with 2,5 volumes of 95% ethanol and 1/15 volume 3M sodium acetate. Pellet was washed with 500ml 75% ethanol, resuspended in DEPC-treated H2O and stored at –80°C. 500 to 1000µg of total RNA for each sample were used for Poly(A)+ RNA isolation with the Micro-Fast Track 2.0 kit (Invitrogen) following the manufacturer’s protocol for yeast mRNA.
2µg of mRNA for each time-point were used for cDNA synthesis by reverse transcription with incorporation of aminoallyl-dUTP (Sigma), then labeled by coupling to N-hydroxysuccinimidyl ester of Cy3 dye (Amerscham) and purified using QIAquick PCR purification kit.
A reference mRNA mix was prepared for each meiotic time course by pooling mRNA corresponding to time 0 h, 2 h and 4 h in the following proportions : 45% / 45% / 10%.
6µl SSC 20X, 1µl SDS, 1µl HEPES and 3µl PolyA were added to 30µl of labelled extracts. Hybridization was performed with an overnight incubation at 63°C in Telechem hybridization chamber.
Cell samples for RNA extraction were taken by centrifugation of culture aliquots (50ml) for 4mn at 3600rpm at 4°C, pellets were resuspended in DEPC-treated H2O, then centrifuged for 4mn at 2500rpm at 4°C. Pellets were immediately frozen in liquid nitrogen and stored at -80°C.
Cells were grown in rich medium (YPD) for 24h, then transferred into presporulation medium (SPS + amino acid supplements) and grown overnight to ~5.107 cells/ml. Cells were then harvested by centrifugation, washed with H2O and resuspended into sporulation medium (1% potassium acetate + amino acid supplements) at a density of 2.107 to 3.107 cells/ml.
Slides were scanned using Genepix 4000B scanner (Axon Instruments) at a resolution of 10µm. Laser power was adjusted to detect the maximum of non-saturating signal.