Adomas et al. BMC Genomics 2010, 11:477 http://www.biomedcentral.com/1471-2164/11/477 there are 1612 such degenerate sequences. Our heuristic search started with a random degenerate 12-mer oligo- nucleotide. Iteratively, this oligonucleotide was randomly mutated and a score was computed as m/(mt * (1 + t + r)), where m, t, and r are the number of hits of the degenerate sequence to the annotated mRNAs, tRNAs, and rRNAs, respectively, and mt is the total number of annotated mRNAs. If a randomly generated number (0- 1) was smaller than the ratio of the new score to the previous, raised to the power of ten, then the new oligo- nucleotide was kept. Otherwise, it was discarded. This procedure was iterated so that the oligonucleotide experienced 2*106 mutations before the search was ter- minated. To ensure convergence upon a globally opti- mal priming sequence, several different random initial sequences were used. Each new oligonucleotide obtained was recorded. From the ranked list, we selected a high-scoring sequence that additionally showed strong binding (GC) in the 3′ end. The MTP primer was designed to be reverse-complementary to the selected dodecanucleotide. Yeast strain and conditions Saccharomyces cerevisiae S288c was grown in synthetic complete medium (SC) at 30°C shaking at 160 rpm until mid log phase (OD600 = 0.4). For nitrogen depletion, cells were collected and resuspended in an equal volume of minimal medium without amino acids or adenine and with limiting concentrations (0.025%) of ammonium sul- fate. Cultures were harvested after 12 h incubation, flash frozen in liquid nitrogen and stored at -80°C [25]. Sample preparation and hybridization to yeast microarrays A set of clones containing 6,188 verified open reading frames (ORFs) from the Saccharomyces Genome Project was amplified by PCR and the DNA was spotted on CMT-GAPS g-aminopolysilane-coated glass slides (Corning, Corning, NY). RNA was extracted and cDNA synthesized as in Townsend et al. [51]. Briefly, two μg of purified mRNA were reverse transcribed using the reverse transcriptase Superscript II (Invitrogen, Carls- bad, CA) for 2 h at 42 C. The reaction was primed with either 0.5 μg oligo-dT primers or 0.25 μg oligo-dT mixed with 0.25 μg of yeast-specific MTP. Amino-allyl- dUTP (Sigma) was incorporated into cDNA along with dNTPs. The cDNA was labeled reciprocally with cyanine dyes and used for hybridization as Townsend et al. [51]. The cDNA from yeast cultured in nitrogen-rich and the cDNA from yeast cultured in nitrogen-depleted medium were reciprocally labeled and competitively hybridized. All replicates originated from independent reverse tran- scription reactions. Page 13 of 16 Data acquisition and analysis Hybridized microarrays were scanned and gridded with a GenePix 4000B microarray scanner (Axon Instru- ments, Foster City, CA), normalized by background-sub- tracted mean-by-mean normalization of well measured spots as in [51,52], and statistically analyzed using a Bayesian analysis of gene expression levels (BAGEL [53]). Fluorescence intensity values were adjusted by sub- tracting background from foreground. A gene was con- sidered well measured if the foreground fluorescence signals were higher than three standard deviations of the distribution of intensities of the background pixels for that gene. Genes were deemed significantly differentially expressed when P ≤ 0.05. To assess the power of the experiments to detect smaller differences in gene expres- sion, the gene expression level at which there was a 50% empirical probability of a significant call (GEL50) was inferred by logistic regression of statistical significance against fold-change as in Townsend [27]. Functional annotation was performed based on the Saccharomyces Genome Database [50] and Kyoto Encyclopedia of Genes and Genomes (KEGG) [54]. Significance of abundantly differentially expressed functional categories was calcu- lated using a Fisher’s exact test. Raw expression data and analysis results are deposited under accession #25 at the Filamentous Fungal Gene Expression Database (FFGED) [36,55] and under accession GSE230003 at the NCBI Gene Expression Omnibus (GEO) database [56]. Transcriptional tag sequencing of yeast in nitrogen-poor conditions Digital Gene Expression (DGE) measurement was per- formed using Tag Profiling with DpnII Sample Prep Kit (Illumina, San Diego, CA). Oligo-dT priming was per- formed as instructed using 2.5 μg of the same total RNA that was extracted from S. cerevisiae for microarrays. For multi-targeted priming, mRNA was purified on Oligo-dT Cellulose Columns (Molecular Research Center, Cincin- nati, OH) and 2 μg were used for reverse transcription with 0.5 μg 5′-amine yeast-specific MTP and 5 mC dNTP 10 mM each (Illumina). The resulting first strand cDNA was cleaned with Microcon-30 microconcentrators and coupled to Sera-Mag® magnetic carboxylate microparti- cles (Seradyn, Indianapolis, IN). cDNAs was immediately subjected to second strand synthesis and sequencing library preparation with the Illumina kit. Samples were then sequenced on an Illumina Genome Analyzer II. Raw sequence reads of transcript tags are deposited under accession #67 at the FFGED [36,55]. Transcript tag analysis The DGE protocol used generates 16-17 nt tags ligated to adapters. RNA sequencing produced reads of 28 bp. After discarding the included adapter sequence, we