Nnat of the Pink module.

<p>The eigenvalue of the Pink module was significantly correlated with nicotine intake (A) but its correlation with food reward earned was not significant (B), suggesting behavioral specificity of the module. Nnat contributed strongly to the expression characteristics of the Pink module (C). The expression pattern of Nnat across the 10 strains measured by PCR showed significant correlation with nicotine intake (D). Strains: BN: Brown Norway, DA: Dark Agouti, F344: Fisher 344, Lew: Lewis, SHR: Spontaneous hypertensive rat, WKY: Wistar-Kyoto. For the F1 hybrids, the two letters representing the initials of the maternal and paternal strains were used.</p

WGCNA detected networks of correlated genes.

<p>(A) Weighted Gene Correlation Network Analysis (WGCNA) was applied to 12,639 genes. A total of 127 modules were identified in AcbS. Each module was named after a unique color assigned by the algorithm. Genes within each module were strongly correlated with each other across all the samples. (B) Modules significantly associated with any of the listed traits are plotted. Twelve modules were associated with stable levels of nicotine intake during last 3 d of SA (when nicotine intakes were stable). Of the 12 modules, nine were negatively correlated with nicotine intake, suggesting their expression in the AcbS protect against voluntary nicotine intake in adolescent rodents.</p

Correlation of nicotine intake with gene expression measured by PCR in AcbS.

<p>A total of 13 genes were selected for validation. The levels of gene expression obtained by RNAseq vs. real-time PCR were correlated with the amount of nicotine intake from the same strains (data reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086214#pone.0086214-Chen3" target="_blank">[29]</a>).</p

Genes of modules correlated with nicotine intake that were implicated in smoking behavior.

<p>A literature mining software, Chilibot <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086214#pone.0086214-Chen5" target="_blank">[48]</a>, was used to search for genes reported to be involved in smoking behavior. Manually curated results confirmed that 15 genes of the Pink module were potentially associated with smoking behaviors. Searching 2 additional modules with similar numbers of genes found only 1 gene potentially associated with smoking behavior.</p

Gene ontology enrichment of the Pink module.

<p>Gene ontology of each module was analyzed using the DAVID function analysis tool. The pink module was enriched with many categories of genes that are involved in synaptic plasticity.</p

Validation of transcriptome sequencing data using real-time PCR.

<p>A total of 13 genes were selected based on the following: strong correlation with the module or nicotine intake; high module membership; strong connectivity within the module. Validation by real-time PCR was conducted using another set of AcbS samples obtained from the same rat strains. The level of gene expression, assayed by transcriptome sequencing vs. PCR across the 10 strains, showed strong correlation (rho  = 0.76 p<0.001).</p