Next generation sequencing analysis of control and KSR1 knockdown CRC cell line translatomes.

KSR1 signaling alters the translational landscape of human CRC cells to support their survival. To determine the effect of KSR1 on translatomes in colon cancer cells, we performed genome-wide polysome profiling. We stably expressed short hairpin RNA (shRNA) constructs targeting KSR1 or a non-targeting control in two K-Ras mutant CRC cell lines, HCT116 and HCT15. We isolated and quantified both total mRNA and efficiently translated mRNAs (associated with ≥ 3 ribosomes) using RNA sequencing. We used Anota2seq to calculate translation efficiency (TE) by comparing the differences in efficiently translated mRNAs to the total transcript of each mRNA. HCT116 and HCT15 control and KSR1 knockdown cells, total RNA and polysomal RNA was extracted using sucrose gradient fraction from three independent experiments for each condition.