Adomas et al. BMC Genomics 2010, 11:477 http://www.biomedcentral.com/1471-2164/11/477 searched for perfect matches of the tag sequence in the S cerevisiae genome. Statistical significance was calcu- lated by estimating the proportion p of tags per gene by maximum likelihood (maximizing pn (1-p)N-n where n is the number of tags observed for the gene and N is the total number of tags for all genes), and comparing the likelihood of the observed numbers of tags with a single maximum likelihood proportion for both samples (p) to the likelihood of the observed numbers of tags with two independent maximum likelihood proportions (p1 and p2, one for each sample). To estimate P-values, negative two times the log likelihood ratio was compared to a chi-square distribution with one degree of freedom. Sta- tistical results for transcript tag analysis are deposited under accession #67 at the FFGED [36,55]. Real Time RT-PCR validation of yeast microarray and RNA sequencing results To validate results of yeast microarrays and RNA sequencing, ten genes were selected whose expression was well measured in the MTP-based experiment, but not well measured or insignificant in the oligo-dT experiment. An equal amount (2 μg) of mRNA, as had been used for microarray profiling, was reverse tran- scribed with Superscript II reverse transcriptase (Invitro- gen, Carlsbad, CA) and 0.25 μg oligo-dT mixed with 0.25 μg yeast-specific MTP or 0.5 μg oligo-dT. Relative transcript abundance was measured using gene specific primers (see Additional File 8) and SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA). The reaction was run on Applied Biosystems 7500 Fast Real- Time PCR System according to manufacturer’s recom- mendations. A gene coding for mitochondrial ribosomal protein of the small subunit (YDR175C), whose expres- sion did not change in the microarray experiment, was used as endogenous control. Transcript levels were cal- culated from triplicates within one plate using the com- parative CT (ΔΔCT) method (ABI application notes, Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR). Induction of Neurospora crassa sexual development Neurospora crassa (mat A) obtained from Fungal Genet- ics Stock Center (strain 2489) was grown on nitrogen- poor synthetic cross medium (SCM) agar plates with 1.5% sucrose as carbon source, at room temperature under natural light. The mycelium was flash frozen by application of liquid nitrogen, then harvested after 18 hours growth. Protoperithecia were similarly harvested, after 6 days. Transcript profiling of Neurospora crassa protoperithecia Total RNA was extracted from homogenized frozen tis- sue and mRNA was purified with TRI REAGENT and Page 14 of 16 Oligo-dT Cellulose Columns (Molecular Research Cen- ter). Reverse transcription, data acquisition and analysis were conducted as for S. cerevisiae. The cDNAs were hybridized [57] to whole-genome-spotted 70-mer N. crassa microarrays [58]. Samples from N. crassa mycelia and protoperithecia were competitively hybridized. Each experiment consisted of a dye-swap and an additional replicate hybridization. Functional classification was based on MIPS Neurospora crassa Genome Database [52] and KEGG [54]. Raw expression data and analysis results are deposited under accession #2 at the FFGED [36,55] and under accession GSE23003 at the NCBI GEO database [56]. Additional material Additional File 1: Number of MTP priming sites in S. cerevisiae genes. Table of the number of MTP priming sites in S. cerevisiae genes Additional File 2: Microarray profiling results comparing S. cerevisiae grown in nitrogen rich (SC) or poor (MM) conditions using oligo(dT) and multi-targeted primers (MTPs) for reverse transcription. Table of microarray profiling results comparing S. cerevisiae grown in nitrogen rich (SC) or poor (MM) conditions using oligo(dT) and multi-targeted primers (MTPs) for reverse transcription. For each priming method for each gene, relative gene expression levels in SC and MM, the additions and subtractions for those levels to demarcate 95% credible intervals, and the p-value for the direction of difference observed are reported. Additional File 3: Comparison of oligo(dT) and MTP priming in terms of number of genes significantly differentially expressed in specific biological processes and metabolic pathways. Table listing biological processes and metabolic pathways with the highest number of genes significantly differentially expressed (P ≤ 0.05) in S. cerevisiae and N. crassa, the number of genes differentially expressed as detected by oligo(dT)-primed and MTP-primed reverse transcription and microarray hybridization, the number of additional genes identified by MTP, and the percent improvement by MTP. Additional File 4: Number of aligned reads recorded for each gene with oligo(dT)-primed and MTP-primed RNA tag sequencing of S. cerevisiae grown in nitrogen rich (SC) or poor (MM) conditions. Table listing each ORF, the number of oligo(dT) primed reads aligning from nitrogen poor (MM) conditions, the number of oligo(dT) primed reads aligning from nitrogen rich (SC) conditions, the number of multi- target primed reads aligning from nitrogen poor (MM) conditions, and the number of multi-target primed reads aligning from nitrogen rich (SC) conditions Additional File 5: Comparison of transcript profiling of S. cerevisiae grown in nitrogen rich (SC) or poor (MM) medium obtained with microarray or RNA tag sequencing using MTP or oligo(dT) primers. Table listing S. cerevisiae ORFs and results of both microarray and RNA tag sequencing by both MTP and oligo(dT) priming. Results listed for each platform and priming methodology are the fold change of MM/SC and the p-value. Additional File 6: Microarray profiling results for N. crassa undergoing sexual development when multi-targeted primers or oligo(dT) primers were used for reverse transcription. Table listing the Broad ID and MIPS ID of well-measured genes in N. crassa protoperithecia (PP) and mycelium (Myc), relative expression levels, additions and subtractions for 95% credible intervals, and p-value, when multi-targeted primers (MTP) or oligo(dT) primers were used for reverse transcription. Additional File 7: Comparison of expression levels of ribosomal genes in Neurospora crassa. Table of ribosomal genes and their fold change in expression in protoperithecia over mycelium, as measured by