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<p>We used Affymetrix Drosophila 2.0 arrays to assess transcript profiles of 3- to 5-d-old flies from the inbred lines. All samples were frozen between 1 and 3 pm. We extracted RNA from two independent pools (25 flies/sex/line), and hybridized 10 g fragmented cRNA to each array. We randomized RNA extraction, labeling and array hybridization across all samples, and normalized the raw array data across sexes and lines using a median standardization.</p>
<p>Each transcript is represented by 14 perfect-match 25-bp oligonucleotides. To identify perfect-match probes with SFPs between the wild-derived lines and the strain used to design the array, we quantified the maximal degree to which the variation between lines could be reduced by partitioning the lines into two allelic classes. We computed the sum of squared deviations from each class mean and expressed their sum as a fraction of the total sum of squares. The smallest fraction across all bipartitions was used to score each probe. We identified 3,136 candidate SFPs with scores 0.1 (a tenfold reduction in the sum of squares). We validated polymorphisms in 20 of 21 of these SFPs by designing primers flanking the SFP and sequencing the PCR products (data not shown).</p>
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