BMC Genomics 2009, 10:295 http://www.biomedcentral.com/1471-2164/10/295 ated with single pass Sanger sequencing [14], Phred sequence trace quality scores were used to remove all tags of low sequence quality (containing bases with a Phred score <20). 21 bp monotags were extracted from both ends of all correctly-sized ditags. Contaminating linker and vector sequences and low-complexity polyA tags were identified and removed by screening a local tag database using formatdb and blast (valid tags in Table 1; <0.06% removed). A total of 819,455 Brassica ESTs were down- loaded on Sept, 15th 2007, from NCBI dbEST http:// www.ncbi.nlm.nih.gov/dbEST/ using the keyword search (Brassica napus [Organism] OR Brassica oleracea [Organ- ism] OR Brassica rapa [Organism] AND EST [Keyword]). About 30% of all Brassica ESTs downloaded from the NCBI dbEST database were produced from seed develop- mental stages. The orientation of all Brassica ESTs was determined by blastx analysis against the TAIR7 A. thal- iana proteome accepting only matches with an e-value of less then 1e-6. Any Brassica ESTs that did not match the A. thaliana proteome using these criteria were removed from the dataset. ESTs that did match the NCBI VecScreen vec- tor database using blastn with pre-set parameters were removed from the analysis. The remaining 834,732 ESTs from NCBI and PBI were annotated based on the align- ment with the A. thaliana proteome and orientated in sense using ReadSeq [36]. Identitag perl scripts were mod- ified so that virtual tags from all positions were extracted from the Brassica ESTs, and the modified Identitag perl scripts were used to create a MySQL database of virtual tags [37]. To differentiate between tags that matched B. napus seed transcripts in sense and in antisense orienta- tion, the oriented sense EST dataset was used in combina- tion with an oriented reverse-complemented EST dataset (antisense EST dataset). For identification of differentially expressed tags the online-tool DiscoverySpace (http:// www.bcgsc.ca/discoveryspace; [38]) was used and signifi- cance calculated according to Audic and Claverie [39]. The online-tools WEGO (http://wego.genomics.org.cn; [40]) and GOEAST (http://omicslab.genetics.ac.cn/GOEAST; [20]) were used for gene ontology (GO) enrichment anal- ysis. Hierarchical cluster analysis with average linkage (UPGMA) and Euclidean distance was performed for GC- RMA processed, normalized data using the software pro- gram HCE 3.0 [41]. Spearman's rank correlations were used to compare Affymetrix chip and LongSAGE [19]. Dif- ferentially expressed genes were identified using the soft- ware program SAM [42]. Cloning of 3'ends of transcripts using SAGE tag primers For validation, the last 17 base pairs of observed Long- SAGE tags (excluding the CATG restriction site) were used as primers for RT-PCR amplification together with anchored oligo-dT primers, as described by Xiao et al. [43], and 2.5 μg total RNA from time-point 35 DAP. cDNA synthesis was performed using ThermoX Reverse Transcriptase (Invitrogen) according to the manufac- turer's instructions. The PCR used 0.5 U Taq Polymerase (Eppendorf) and 250 nM of each primer, with 2 min at 95°C followed by 30 cycles of 1 min 95°C, 1 min 50°C, 2 min 72°C and final extension at 72°C for 10 min. Strand-specific Real-time RT-PCR Total RNA was extracted as described above and addition- ally purified using RNAeasy columns (Qiagen). DNase digestion was performed using RQ1 RNAse-free DNAse (Promega) according to manufacter's recommendations. cDNA was synthesized after denaturation for 10 min at 70°C using 1 μg of total RNA, 100 nM of gene and strand- specific reverse primer and Omniscript Reverse Tran- scriptase (Qiagen) according to the manufacturer's recom- mendations in a total volume of 10 μl. After the cDNA synthesis reaction was finished the enzyme was inacti- vated for 15 min at 95°C. Real-time PCR was performed using a 7500 Fast Real-time PCR system (Applied Biosys- tems) with a total volume of 10 μl using 1 μl of cDNA (1:10 diluted), 5 μl of Power SYBR Green PCR master mix (Applied Biosystems) and 100 nM each of the forward and reverse primers. Amplification was for 2 min at 50°C, 10 min at 95°C followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. Controls included PCR amplification reactions using cDNA preparations without adding the reverse transcriptase enzyme to check for genomic DNA contamination, and PCR amplification reactions without adding reverse primers during cDNA synthesis to check for a loss in strand-specificity by RNA self priming leading to primer-independent cDNA synthesis [44]. Primers were designed by using Brassica ESTs that were matched using blastx at an e-value cut-off of 1e-6 with selected A. thaliana proteins. All matched Brassica ESTs were assembled using the software CAP3 [45] and edited manually. Alignments of coding DNA were constructed from aligned amino acid sequences using the software RevTrans [46]. Contigs from these assemblies were used for primer design using the software PrimerExpress (Applied Biosystems). Primer pairs used for sense transcript detection were Napin_for (5'ACGAGCTCCACCAGGAAGAG-3') and Napin_rev (5'- ACGGCTTTGGATGCTCCTT-3'), which amplify position 320 to 383 relative to the coding region of gene napB [GenBank:X14492], and Napin-related_for (5'-AACAAG- CAGCCAAGTCAGTTAGG-3') and Napin-related_rev (5'- GATGCGGGTGGACTGGAAT-3') which amplify position 323 to 384. For antisense transcript detection the reverse complemented sequences of the primers were used. Expression fold-changes were calculated relative to the endogenous reference gene cyclophilin (GenBank acces- sion M55018) according to the 2-ΔΔCt method using three replicates [47]. Authors' contributions RS conceived the project and WF provided the plant mate- rial. CO and BH generated the SAGE libraries. CO adapted and applied bioinformatic tools for analysis of LongSAGE Page 14 of 16 (page number not for citation purposes)