Additional file 1:

Verification of SSH procedure. (A) To estimate the efficiency of subtraction, the abundance of actin transcripts was analyzed by qPCR using unsubtracted (white bars) and subtracted cDNAs (red bars) from forward and reverse subtractions as templates. The result demonstrated that the abundance of actin, a non-specifically expressed housekeeping gene, was greatly decreased in subtracted samples. The unsubtracted sample is a control that is integral to the subtraction process, so it was subjected to the same dilutions and amplifications as the corresponding subtracted sample. (B) Differential expression of a random population of cloned genes (126 clones) was measured by filter hybridization as described in Methods. The graph shows the normalized hybridization signal intensities of each spot on the y axis for stages 6-7 (S6-7) and on the x axis for stages 2-3 (S2-3). The intensity values of 76% of the clones were at least 2-fold higher in S6-7 than in S2-3. (C) Virtual northern blots analysis using cDNA from syncytial blastoderm (S2-3) and gastrula (S6-7). Labeled probes corresponded to 10 clones that were four-fold overexpressed in gastrula compared to syncytial blastoderm in panel B. In each case, stronger hybridization signals were obtained with the gastrula cDNA, confirming the results from the microarray assays. Signal intensities were normalized to that of the actin gene.

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Additional file 2:

Functional composition of the subtracted library. Comparison of gene ontology (GO) categories between the subtracted library and the whole D. melanogaster genome based on 11 Molecular Functions (A) and 14 Biological Processes (B). The percentage (x axis) of each GO term (y axis) indicates the representation of each category within the subtracted library (red bars) or the whole genome (blue bars).

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Additional file 3:

Genes up-regulated during embryo development. For each protein-coding gene and ncRNAs, the table displays the CG number and name, which links to FlyBase [66]. The gene ontology (GO) terms are: Molecular Function, Biological Process and Cellular Component. Homology/Protein Domain: protein domains that were identified using bioinformatics tools. Cluster number: the number of the group in which each gene was contained after the hierarchical cluster analysis. In situ Pattern: pattern of spatial expression described in our present work. Literature: a list with references citing studies in which the genes were found to be differentially expressed during D. melanogaster development. For intergenic regions, the table displays the clone ID, GenBank accession number, and genomic location.

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Additional file 4:

Primers used for qPCR and conventional PCR reactions. The table contains primer names, amplicon sizes, annealing temperatures (Tm) and primer sequences.

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