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Specifically, RNA-seq data of 566 tumor samples and 37 adjacent normal tissue were downloaded for further analysis. Raw reads were trimmed to remove TruSeq adapter sequences and primers using Trim Galore! and aligned to hg38 assembly using STAR 2.5.1b [17]. Alignments were filtered to retain only reads mapping uniquely. For each sample, the percentage of reads mapping to annotated exons was calculated using featureCounts [18] and normalized by total number of reads mapped to exons. Reads that did not map to any of the annotated exons were discarded. For each cell line, the average of the three replicates was used. The heatmap was generated using the heatmap.
The RNA-seq data was generated with Illumina Nextseq. Reads were trimmed to remove TruSeq adapter sequences and primers using Trim Galore! and aligned to hg38 assembly using STAR 2.5.1b [17]. Alignments were filtered to retain only reads mapping uniquely. For each sample, the percentage of reads mapping to annotated exons was calculated using featureCounts [18] and normalized by total number of reads mapped to exons. Reads that did not map to any of the annotated exons were discarded. For each cell line, the average of the three replicates was used. The heatmap was generated using the heatmap.2 function in R. The heatmap.2 function was also used to display the expression values (log2 read count) of the top 10 most up-regulated genes in the OSU-CCC cell lines in comparison to the C13-E2 cell line. The gene expression profiles of OSU-CCC and C13-E2 cell lines were clustered using the Cluster 3.0 and visualized using Java TreeView [19].
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