Bgi500 platform

The total RNA of each species was extracted from one plant using the EASY spin Plus Complex Plant RNA Kit (Aidlab, Beijing, China). The RNA purity, concentration, and integrity were evaluated using a Nanodrop (Thermo Fisher Scientific, Cleveland, OH, USA), Qubit 3.0 (Invitrogen, Waltham, MA, USA), and Agilent2100 (plant RNA Nano Chip, Agilent, Santa Clara, CA, USA), respectively. The ribosome RNA was depleted by the RiboZero Magnetic Kit (Epicenter, Madison, WI, USA), and a library was then built using a TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA). Furthermore, RNA-seq was conducted by Beijing Genomics Institution (BGI) using the BGI500 platform set 100 bp for the length of paired-end (PE) reads, by Mega Genomics (MG, Beijing, China) using an Illumina HiSeq X-Ten platform (PE 150 bp), or by Berry Genomics Corporation (BGC, Beijing, China) with an Illumina NovaSeq 6000 platform (PE 150 bp).

SW620 and colon26 cells were treated as described with CXD101 or DMSO as a negative control. Total RNA (triplicates unless otherwise stated) was isolated using Direct‐zol RNA MiniPrep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's instructions. RNA sequencing was performed by BGI Genomics (Beijing, China). Briefly, an Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit; Santa Clara, CA, USA) was used for RNA sample quality control purposes (RNA concentration, RIN value, 28S/18S and the fragment length distribution). mRNAs were isolated from total RNA using the oligo(dT) method. Then, the mRNAs were fragmented, and first‐strand/second‐strand complementary DNAs (cDNAs) were synthesised. cDNA fragments were purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. Subsequently, the cDNA fragments were linked with adapters. Those cDNA fragments with suitable size were selected for the PCR amplification. Agilent 2100 Bioanalyzer and ABI StepOnePlus Real‐Time PCR System were used in quantification and qualification of those libraries. The RNA sequencing was carried out using Illumina HiSeq Platform (SW620) or BGI500 platform (colon26 in vitro and in vivo).