Transcriptomic analysis of immortalized oral keratinocytes (n=2) and oral squamous cell carcinoma cells (n=5)

Due to betel nut chewing habit, oral cancer is the fourth leading cause of cancer death among Taiwanese men. In addition, oral cancer is the most common malignancy observed in men with 25-44 years old, alerting an urgent need to curb this disease. It is hypothesized that betel nut ingredients exhibit carcinogenic effects on the oral epithelium and alter local immune system. As a step to identify potential oncogenic gene fusions in Taiwanese oral cancer cells, total RNAs of two immortalized oral keratinocytes (CGHNK2, -K6) and five oral squamous cell carcinoma cell lines (C9, OC3, OEC-M1, TW2.6) were subjected to RNA-sequencing analysis, followed by an in-house bioinformatic pipeline for fusion gene detection. Total RNA was extracted from specified cell lines before reaching confluency , and the quality of the RNA was assessed with the Agilent Bioanalyzer. Transcriptome libraries from the mRNA fractions were generated following the RNA-Seq protocol (Illumina). Each sample was sequenced in a single lane with the Illumina Genome Analyzer II with an 80-nt read length. Paired-end transcriptome reads passing our filters were mapped to the human reference genome (hg19) using Tophat2. Gene expression profiles were estimated by Cufflinks from SAM/BAM files generated in the previous step. Independently, sequence alignments were processed to nominate gene fusions by using ChimerScan (PMID: 21840877).