order partial correlation was used to detect the interaction between trans-regulated transcripts and cis-regulated candidate genes conditioned on the genotype (marker rs8242481 at 175.058 Mb). If x, y and z are trans-regulated transcripts, cis- regulated transcript, and genotype in the QTL, respectively, then the first order partial correlation coefficient is calculated as— rxy{rxzryz rxy:z~ rﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1{r2xz 1{ry2z where rxy can be either Pearson correlation or Spearman’s rank correlation between x and y. We employed the Spearman’s rank correlation because the expression levels of many transcripts do not follow a normal distribution. The significance of a partial correlation with n data points was qﬃnﬃ{ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ assessed with a two-tailed t test on t~r 1{2{r2 k*tn{2{k where r is the first order correlation coefficient, and k is the number of variables on which we are conditioning. Immunocytochemistry Cultured hippocampal neurons from male B6 mice, prepared as described in Schikorski et al. [111] and cultured for 23 days, were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in HEPES buffered saline (pH 7.2) for 15 min. Cell membranes were permeabilized with 0.1% triton X-100 and unspecific binding sites were quenched with 10% BSA for 20 min at room temperature (RT). Neurons were incubated with a polyclonal anti-FMN2 antibody (Protein Tech Group, www.ptglab.com) diluted to 0.3 mg/ml at RT overnight. An anti-rabbit antibody raised in donkey (1:500, Invitrogen; http://www.invitrogen.com) conjugat- ed with the fluorescent dye Alexa488 was used for the detection of the first antibody. All regions of interest were photographed with identical illumination and camera settings to allow for a direct comparison of the staining in labeled and control neurons. Fmn22/2 and Fmn2+/+ Microarray Analysis The Fmn22/2 mice were generated using 129/SvEv (now strain 129S6/SvEvTac) derived TC-1 embryonic stem cells. Chimeric mice were backcrossed to 129/SvEv [70]. The Fmn2-null and littermate controls are therefore coisogenic. To validate the isogenicity of regions surrounding the targeted locus [112], we genotyped the Fmn2+/+, Fmn2+/2, and Fmn22/2 mice using ten microsatellite markers located on, and flanking Fmn2 (markers distributed from 172 Mb to 182 Mb). These markers are D1Mit455, D1Mit113, D1Mit456, D1Mit356, D1Mit206, D1Mit355, D1Mit150, D1Mit403, D1Mit315, and D1Mit426. With the exception of a marker at Fmn2 (D1Mit150), all alleles in null, heterozygote, and wildtype animals were identical. RNA was isolated from whole brain samples of Fmn2+/+ and Fmn22/2 mice, and assayed on Illumina Mouse-6 array slides (six samples per slide). We compared five samples from Fmn22/2 nulls, and five samples from Fmn2+/+ wildtype. Equal numbers of each genotypes were placed on each slide to avoid batch confounds. Microarray data were processed using both raw and rank invariant protocols provided by Illumina as part of the BeadStation software suite (www.illumina.com). We subsequently log-transformed expression values and stabilized the variance of each array. To identify genes with significant expression difference between the Fmn22/2 and Fmn2+/+ cases, we carried out two-tailed t-tests and applied a Bonferroni correction for multiple testing, and selected probes with a minimum adjusted p-value,0.05. QTL Hotspot on Mouse Distal Chromosome 1 Bioinformatics Tools Classical QTLs counts are based on the April 2008 version of Mouse Genome Informatics (MGI: www.informatics.jax.org) [113]. Search for tRNAs was done using tRNAscan-SE 1.21 (http://lowelab.ucsc.edu/tRNAscan-SE/) [65]. GO analysis was done using the analytical tool DAVID 2007 (http://david.abcc. ncifcrf.gov/) [114]. Overrepresented GO terms were identified and statistical significance of enrichment was calculated using a modified Fisher’s Exact Test or EASE score [115]. We used the Allen Brain Atlas to analyze expression pattern in the brain of young C57BL/6J male mice (www.brain-map.org) [85,116]. Control for Non-Syntenic Association and Paralogous Region In RI strains, non-syntenic associations can lead to LD between distant loci [89,106]. In the BXDs, we detected such non-syntenic associations between markers in Qrr1 and markers on distal Chr 2 and proximal Chr 15. As a result of these associations, some transcripts that have strong cis- or trans-QTLs in Qrr1 tend to have weak LOD peaks, usually below the suggestive threshold, on distal Chr 2 and proximal Ch15. However, there is no bias for genes located in these intervals in LD with Qrr1 to have trans-QTLs in Qrr1. The Qrr1 segment has been reported to have paralogues on mouse Chrs 1 (proximal region), 2, 3, 6, 7, 9, and 17 [117,118]. We examined if the trans-QTLs in Qrr1 are of genes located in these paralogous regions. However, genes located in the paralogous regions are not overrepresented among the trans-QTL. Supporting Information Table S1 Number of classical QTLs in Qrr1 and in hundred other chromosomal intervals. Found at: doi:10.1371/journal.pgen.1000260.s001 (0.23 MB DOC) Table S2 Transcripts of genes associated with seizure or epilepsy that have trans-QTLs in Qrr1p near the seizure susceptibility QTL. Found at: doi:10.1371/journal.pgen.1000260.s002 (0.05 MB DOC) Dataset S1 Precision of Cis-QTLs in Qrr1. Found at: doi:10.1371/journal.pgen.1000260.s003 (0.13 XLS) Dataset S2 Gene ontology analysis of transcripts that map to Qrr1p and Qrr1d in the BXD hippocampus dataset. Found at: doi:10.1371/journal.pgen.1000260.s004 (0.03 MB XLS) Dataset S3 tRNAs in Qrr1d. Found at: doi:10.1371/journal.pgen.1000260.s005 (0.09 XLS) Dataset S4 Partial correlation analysis. Found at: doi:10.1371/journal.pgen.1000260.s006 (0.04 XLS) MB MB MB Acknowledgments We thank Drs. John K. Belknap and Robert Hitzemann for access to the B6D2F2 datasets. We thank Drs. Gerd Kempermann and Rupert Overall and members of the Hippocampus Consortium for access to their dataset, and we thank Dr. Glenn Rosen for access to the BXD striatum datasets. We thank Drs. Ivan Rusyn and David Threadgill for access to the BXD UNC liver dataset, and we thank Drs. Gerald de Haan and Michael Cooke for access to the BXD hematopoietic stem cells dataset. We thank Drs. Erwin P. Bottinger and Kremena V. Star for access to the BXD kidney PLoS Genetics | www.plosgenetics.org 15 November 2008 | Volume 4 | Issue 11 | e1000260