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Supplementary Figure 1: (A) Flow diagram describing the microarray data analysis. (B) Box plot depicting raw expression data for 47,323 gene
probes post log transformation and quantile normalization expression for 26 Healthy Control (HC) and 22 Bipolar (BP) brain samples. (C) Quality
Inspection - covariance-based Principal Component Analysis (PCA) scatterplot depicting the absence of both cohort-level and gross-level outliers.
(D) Quality Inspection - correlation-based heat map showing the absence of both cohort-level and gross-level outliers. (E) Noise Analysis - XY plot
describing the data to have a LOWESS fit (CV~mean) by disease class (i.e., BP vs HC). The resulting curves plotted with mean expression (i.e.,
signal) on the x-axis and coefficient of variation (i.e., noise) on the y-axis. Plot was used to identify the mean expression value where the linear
relationship with noise, from right to left, is grossly lost (vertical dashed line). This mean expression value (7.75) was defined as the signal
threshold for the data and all expression values less than the threshold floored to equal the threshold. Gene probes not having at least one
expression value across all samples greater than the signal threshold were discarded as non-informative.
Supplementary Figure 2: PCA plots of NISC1 and NISC2 reveal separation between DE genes in BP (red dots) and controls (blue dots). Principal
component 1 (PC1) is shown on X-axis, PC2 on Y-axis and PC3 is represented on Z-axis.
Supplementary Figure 3: Raw distribution of read counts in NISC1. “bp” indicates bipolar disorder cases and “ctl” indicates psychiatrically
healthy controls. Each plot shows raw count values for each of the 9 specimens in the NISC1 sample, (4 cases and 5 controls). Overall range of
counts is 0 through 4 * 10^7 for all specimens, with a median around 300. Total of 25,017 genes were taken into account for each specimen.
Supplementary Figure 4: Raw distribution of read counts in NISC2. “bp” indicates bipolar disorder cases and “ctl” indicates psychiatrically
healthy controls. Each plot shows raw count values for each of the 12 specimens in the NISC2 dataset, (6 cases and 6 controls). Overall range of
counts is 0 through 8 * 10^7 for all specimens, with a median around 300 counts. Total of 25,017 transcripts taken into account for each
specimen.
Supplementary Figure 5a: An overview of sequencing analysis pipeline
Supplementary Figure 5b: GWAS enrichment test
GWAS
Enrichment test
Step1: Count total # of SNPs (N) assigned to all genes in a GO term
Step 2: Count # of SNPs (n) with GWAS p-value<0.05
Step3: Randomly draw N SNPs (Nr) from GWAS data 10,000 times
Step 4: Count # of SNPs with GWAS p-value<0.05 in Nr
Supplementary Figure 6: Box plots for all genes differentially expressed at FDR <5% in the gene-level meta-analysis (shown separately by
sample, and arranged alphabetically by HUGO gene symbol). Normalized count data is shown on the y-axis for both the control (ctl) and bipolar
(bp) groups. Box plots show the lower and upper extremes and the median for each group.
Supplementary Figure 7: NISC1 heatmap for genes (gene-level) with p<0.05 (left). Hierarchical clustering of 919 mean centered differentially expressed
genes between bipolar disorder cases and control samples in NISC1 sample. The first 5 columns show control data; the last 4 columns show bipolar
disorder case data. Red and green clusters in the dendrogram represent genes more highly expressed in bipolar disorder cases than controls. Blue
clusters represent genes more highly expressed in controls than cases. Gene profile plots (right) represent the same clustered genes as the heatmap.
Supplementary Figure 8: NISC2 heatmap for genes (gene-level) with p<0.05 (left). Hierarchical clustering of 1181 mean-centered differentially
expressed genes between bipolar disorder cases and control samples in NISC2 sample. The first 6 columns show control data; the last 6 columns
show bipolar disorder case data. Red clusters in the dendrogram represent genes more highly expressed in bipolar disorder cases than controls.
Green and blue clusters represent genes more highly expressed in controls than cases. Gene profile plots (right) represent the same clustered
genes as the heatmap.
Supplementary Figure 9: Box plots for all transcripts differentially expressed at FDR <5% in the transcript-level meta-analysis (shown separately
by sample, and arranged alphabetically by HUGO gene symbol). Normalized count data is shown on the y-axis for both the control (ctl) and
bipolar (bp) groups. Box plots show the lower and upper extremes and the median for each group.
Supplementary Figure 10: NISC1 heatmap for transcripts (transcript-level) with p<0.05 (left). Hierarchical clustering of 1817 mean-centered
differentially expressed transcripts between bipolar disorder cases and control samples in NISC1 sample. The first 5 columns show control data;
the last 4 columns show bipolar disorder case data. Red and green clusters in the dendrogram represent transcripts more highly expressed in
bipolar disorder cases than controls. Blue clusters represent transcripts more highly expressed in controls than cases. Transcript profile plots
(right) represent the same clustered genes as the heatmap.
Supplementary Figure 11: NISC2 heatmap for transcripts (transcript-level) with p<0.05 (left). Hierarchical clustering of 3255 mean-centered
differentially expressed transcripts between bipolar disorder cases and control samples in NISC2 sample. The first 6 columns show control data;
the last 6 columns show bipolar disorder case data. Blue clusters in the dendrogram represent transcripts more highly expressed in bipolar
disorder cases than controls. Red and green clusters represent transcripts more highly expressed in controls than cases. Transcript profile plots
(right) represent the same clustered genes as the heatmap.
Supplementary Figure 12: Technical validation of selected genes by digital barcoding (Nanostring). Scatterplot of Log 10 normalized counts
(NISC1 sample) for RNA-seq (x-axis ) and Nanostring (y-axis). Colored rings represent the 50% confidence ellipses for each of the 41 genes tested.
Genes whose expression was more highly correlated across the 2 assays produce ellipses that are more closely parallel to the diagonal ( ).