Next generation sequencing facilitates quantitative analysis of oncogene induced transcriptomes

Expression of CDC25A, CCNE1, or MYC in MDA-MB-231, BT549, HCC1806, RPE1-TP53wt, and RPE1-TP53mut cells was induced using 1 µg/mL of doxycycline. Cells were harvested and frozen at -80°C at 48 hours and 120 hours after doxycycline induction. Next, RNA was isolated using the mirVANA kit (Ambion, AM1561). To determine RNA quality, RNA was separated by electrophoresis on microfluidic sipper chips and detected by fluorescence (LabChip GX, Caliper LifeSciences). RNA Quality Scores (RQS) were based on electropherogram features (total and fast region areas, 28S, and 18S height), and ranged from 0-10. Only samples with RQS scores above 5 were included for analysis. To generate cDNA libraries suitable for next-generation sequencing (NGS), the QuantSeq RNAseq 3' mRNA kit (Lexogen) was employed. Briefly, the poly-A tail from total RNA was bound to oligo-dT primers to synthesize the first cDNA strand. After RNA removal, a second cDNA strand was synthesized by random priming. The double-stranded cDNA library was purified, and PCR amplified with Illumina sequencing adapters. The libraries were sequenced with 65 base-pair reads on a NextSeq 500 sequencer (Illumina) and generated 7.2 to 19.8 million of reads per sample. Expression of CDC25A, CCNE1, or MYC in MDA-MB-231, BT549, HCC1806, RPE1-TP53wt, and RPE1-TP53mut cells was induced using 1 µg/mL of doxycycline. Cells were harvested and frozen at -80°C at 48 hours and 120 hours after doxycycline induction.