GSE133201
Gene expression profiling of two subclones of HN9-S and HN9-R isolated from patient-derived HN9 cancer cells
Purpose: The goals of the study are to understand phenotypically different characteristics of two single clones, HN9-R and HN9-S isolated from primary Head and Neck Carcinoma HN9 and to compare transcriptome profiling to genome-wide analytic approach performed by next-generation sequencing (NGS) analysis. Methods: mRNA profiles of two single clones, HN9-R and HN9-S, were generated by sequencing using Illumina NextSeq 500 software. Sequenced reads were trimmed for adaptor sequence then mapped to hg19 whole genome using STAR (version 2.5.0) with parameters -outFilterIntronMotifs RemoveNoncanonicalUnannotated. Fragments Per Kilobase of exon per Megabase of library size (FPKM) were calculated by Cufflinks packages (version 2.2.1). Differentially expressed genes were detected using Cuffdiff (version 2.2.1) against Ensemble genes. Results: We find total 337 DEGs (differentially expressed genes) between HN9-R and HN9-S (FPKM value of > 1, log2. Fold-change > 2, and Padj < 0.05) and that there were 178 and 159 genes of significantly increased or reduced transcripts, respectively. The level of gene expression was validated by quantitative real-time PCR using SYBR Green assay and western blotting analysis. Conclusion: The results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cells. This study provides a framework for the dissection of biological functions towards characterization of two sub-clones co-existed within a primary cancer cells. Overall design: Gene expression profiles of two clones (HN9-R and HN9-S) isolated from Head and Neck Cancer cells HN-9 were generated by sequencing, in triplicate, using Illumina NextSeq 500.