de Jong S et al. BMC Genomics 2010, 11:20 http://www.biomedcentral.com/1471-2164/11/20 Database (NCBI 37, http://phenome.jax.org/pub-cgi/phe- nome/mpdcgi?rtn=docs/home), comparing SNPs between A/J and C57BL/6J. Total SNPs were counted and the number of synonymous and non-synonymous coding SNPs was recorded. In addition, Illumina 50-mer probe sequences were blasted (NCBI megaBLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi) against the FASTA sequences, including the flanking sequences (obtained via NCBI SNP 37.1, http://www.ncbi.nlm.nih. gov/sites/entrez, of SNPs in exons or UTR regions. The significance of the differences between SNP counts was tested using a Fisher’s exact test for count data with a threshold of p < 0.05. Cis effects were checked in the eQTL database generated with the heterogeneous stock of the Welcome Trust Case Control Consortium http:// gscan.well.ox.ac.uk/. The significance of the differences between counts was tested using a Fisher’s exact test for count data with a threshold of p < 0.05. In addition, sequencing of probe sequences not containing a known SNP was performed. Primers were designed in NCBI’s Primer BLAST http://www.ncbi.nlm.nih.gov/tools/pri- mer-blast/ to generate a PCR product. Sequencing was performed according to standard protocols on an ABI 3730 (Applied Biosystems) sequencer. A list of probes and primers can be found in Additional File 5. Gene ontology analysis Ontological enrichment for functional categories was studied using Ingenuity Pathways Analysis (Ingenuity® Systems, http://www.ingenuity.com). We obtained a bar plot of functional categories with at least one module enriched at the p < 10e-04 level. Additional file 1: Eigengene significance for mouse strain and brain region. The module eigengene is a single representative expression profile for each sample, based on the first principal component of that module. The first bar plot shows that the Magenta module eigengene is significantly correlated with strain. This indicates that this module is significantly enriched for genes differentially expressed between A/J and C57BL/6J. The second barplot shows that the Red and Pink module eigengenes are significantly correlated with brain region. This indicates that these modules are significantly enriched for genes differentially expressed between amygdala and hippocampus. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 20-S1.PDF ] Additional file 2: Content of the Magenta, Pink, and Red modules. Tables contain probes in Magenta (table 1), Pink (table 2) and Red (table 3) modules. Illumina probe ID, Gene Symbol, Chromosome and expression differences AJ versus C57BL6/J and hippocampus versus amygdala are given. Asterisks in the Probe ID column denote probes containing a known SNP between A/J and C57BL/6J. The last column denotes the cis-regulated genes according to the WTCCC Heterogeneous stock database http://gscan.well.ox.ac.uk/, with and without known SNPs between included strains. Expression data is transformed (variance stabilizing method) and normalized (robust spline) yielding values comparable to LOG2 values. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 20-S2.XLS ] Page 12 of 14 Additional file 3: Magenta, Pink and Red module membership for all genes. Table contains the correlation between the eigengenes (i.e. ‘module membership’) for the Red, Pink and Magenta modules with expression values for all 13,627 detected genes on Illumina array. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 20-S3.XLS ] Additional file 4: Network reconstruction on subsets of samples. Networks constructed per strain or per brain region identify distinct modules of co-expressed genes. Dendrograms were produced by average linkage hierarchical clustering of genes using the topological overlap measure. Modules of co-expressed genes were assigned colors corresponding to the branches indicated by the horizontal bar beneath each dendrogram. The color code of the previous module definition (using all samples of both strains) was used to assess preservation. The Red and Pink module are preserved in networks constructed on just A/J (n = 17) or just C57BL/6J samples (n = 18). The Magenta module is found in the networks constructed on just amygdalar (n = 17) or just hippocampus samples (n = 18). Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 20-S4.PDF ] Additional file 5: Additional polymorphisms in probe sequences. Sequencing was performed for the probes not containing a known SNP. Probes in the Magenta module showing the highest differential expression between C57BL/6J and A/J were selected. The first and second columns contain the Illumina probe ID and gene symbol of the corresponding target gene. The third column shows the probe sequence for these probes. The forward and reverse primers used are shown in column 4 and 5. The last column indicates whether a SNP (or indel) was found in the probe sequence, which is also indicated in the probe sequence. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 20-S5.XLS ] Additional file 6: Ingenuity Pathway Analysis for the modules of interest. Ingenuity pathway results for genes in the Red (table 1), Pink (table 2) and Magenta (table 3) modules. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 20-S6.XLS ] Additional file 7: Strain * brain tissue interaction effects. A linear model analysis was performed to assess possible interaction effects of brain tissue * strain for all 13627 probes. For this, the R package Limma was used, constructing a linear model with interaction term. Significance was assessed using FDR correction. After this correction, 6 genes showed significant interaction (adjusted p-value < 0.01). The first three columns of the table contain the Illumina Probe ID, Gene Symbol and chromosome. Column four indicates the module in which the probe was grouped using WGCNA analysis. Linear model statistics are given in column five and six. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 20-S7.XLS ] Abbreviations SNP: Single Nucleotide Polymorphism; CNV: Copy Number Variation; GABA: g-Aminobutyric acid; WGCNA: Weighted Gene Coexpression Analysis. Acknowledgements We would like to thank Hugo Oppelaar, Cathy Fernandes and Heidi Lesscher for practical assistance and advice. We also thank Jake Lusis and Peter Burbach for helpful discussions and Jackie Senior for critically reading the manuscript. The study was supported by travel grants from the Van Walree Fund, the Royal Netherlands Academy of Arts and Sciences, and the Utrecht University California Research Cooperation Fund to SDJ, and research funding from the National Institutes of Health to RAO.