GSM5051911: u1: untreated cells, replicate 1; Homo sapiens; RNA-Seq
The HT29 colon cancer cell line were incubated with CPT-CEF as mentioned in our previous study(Krishnan et al., 2017). Then, the total RNA was extracted from the cells using RNeasy mini kit according to recommendation from the manufacturer (Qiagen; Hilden Germany). Samples with RNA integrity (RIN) value more than 7 and high purity (A260/A280 ratio = 2) would be used for downstream experiment. The Bioanalyzer 2100 system (Agilent Technologies; CA, USA) was used to measure RNA integrity whereas the NanoDrop 2000 Spectrophotometer (Thermo Scientific; MA, USA) was used to measure the A260/A280 ratio. Library preparation was performed using the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (New England Biolabs, MA, USA) according to the manufacturer's protocol. The isolated RNA was further enriched to obtain mRNA. To do this, total RNA was incubated with 50 µL of oligo dT beads and binding buffer at 65°C for 5 mins in a PCR tube. The tubes were then placed onto a magnetic stand to capture the beads. The contents of the tubes were washed several times to remove residual rRNA and other RNA types. Then, the beads were mixed with 50 µL of Tris-buffer and incubated in a thermal cycler at 80°C for 2 mins, followed by a cooling step to a temperature of 25°C before elution.