Experimental Protocols

For the histone H4 depletion experiment, duplicate cultures of strain UKY403 were switched from galactose to glucose media, harvested, and labeled cRNA prepared from purified mRNA was hybridized to Affymetrix GeneChip arrays (20-22). To control for the effects of the galactose to glucose shift, duplicate cultures of strain MHY308, which contains a wildtype histone H4 gene, were analyzed in parallel.

For the SIR2, SIR3, and RAP1 deletion mutants, two independent experiments were performed for each wild type versus mutant comparison. Individual mRNA levels were scored if the computer algorithm analyzing the scanned results returned a "Present" call in both the two wild type and the two mutant expression profiles for that gene or if the expression levels of that gene changed in the same direction and were greater than background levels in both wild type and mutant comparisons (20).

The protocols below describe how cells were grown and harvested, how total RNA and polyA RNA was prepared. All subsequent steps were carried out exactly as described in the Affymetrix product information supplied with the Genechips (the leaflets are part numbers 700187 Rev 1 and 700163 Rev 1 5/98).

Cell Harvesting
Total RNA Preparation
PolyA mRNA Preparation