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text: Protocols: Human CRC tissues were harvested by surgery. Tissues containing 1 x 106 cells were stored in 1 mL TRIZOL (Thermo Fisher Scientific) and ground in liquid nitrogen. Next, 100 μL chloroform was added, and the cells were fully mixed and centrifuged at the highest speed at 4 °C for 10 min. The supernatant was transferred into a new tube, isopropanol was added to the same volume, and cells were centrifuged at the highest speed at 4 °C for 10 min. The precipitate was washed with 75% cold ethanol and dissolved in DEPC water. The concentration and quality of RNA were measured using a Nanodrop 2000 (Thermo Fisher Scientific) and an Agilent Bioanalyzer 2100 (Agilent, USA). A total of 2 μg of RNA from each group was used for library preparation using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, USA) following the manufacturer’s instructions

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