nucleic acid extraction protocol
To extract total RNA yeast cells were lysed by vortexing with glass beads in the presence of hot acid phenol. RNA was then cleaned by phenol:chlorophorm extraction followed by ethanol precipitation. polyA RNA isolation with subsequent cDNA synthesis with dUTP incorporation and in presence of actinomycin D.
nucleic acid labeling protocol
Fragmentation and biotinylation with Affymetrix GeneChip WT Terminal labeling kit.
array scanning and feature extraction protocol
Scanner type: GeneChip 3000 7G. Scanning software: default software
nucleic acid hybridization to array protocol
Modified tiling array Affy target Hybridization with GeneChip control oligoB2, Affymetrix.
For YPD_BPS and YPMalt:Grow single colony in 3 ml YPD for the day at 30C. Dilute in 100 ml of desired medium with starting OD600=0.0019. Grow to OD600=1.0 at 30C. Harvest cells for total RNA extraction.for YPD_Rapa/YPE:Grow single colony overnight in 5 ml YPD at 30C. Dilute in 30 ml of desired medium with starting OD600 = 0.25. Grow the culture to OD600=0.9 for the day. Incubate the day culture in 400 ml of desired medium at 30C overnight. Harvest cells at OD600 at 1.0 for total RNA extraction.