Hiseq 2500

Total RNA was isolated from 1 × 10⁷ cells using the mirVana™ miRNA ISOlation Kit (Ambion, Austin, TX, United States) following the manufacturer's instruction. 1 μg of total RNA was used to prepare standard RNA-seq libraries (TruSeq Stranded Total RNA kit with Ribo-Zero Gold, Illumina). RNA integrity was validated for size using the Agilent 2100 Bioanalyzer (Agilent Technologies, CA, United States) and sequenced by a 2 × 125 bp paired-end sequencing module on an Illumina HiSeq 2500 (Oebiotech, Shanghai, China). The criteria for differential gene expression included a fold change ≥2 between compared groups and statistical significance at P < 0.05.

The total mRNA was obtained from HCT116 cells treated with either oxaliplatin or GSK-J4 for 48 h. After total RNA extraction, samples were processed by Hi-Seq 2500 by OE Biotech. Gene expression levels were analyzed by the software HTSeq-count and Cufflinks (version 0.6.1) (Roberts et al., 2011 (link); Anders et al., 2015 (link)). All differentially expressed genes generated by the software DESeq (version 1.34.1) were then analyzed by the DAVID bioinformatics platform to enrich biological terms.

Total RNA from cecal samples in each group was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The purity of RNAs was checked using a NanoPhotometer^® spectrophotometer (IMPLEN, CA, USA). The concentration and integrity of RNAs were determined using the Qubit^® RNA Assay Kit in Qubit^® 2.0 Flurometer (Life Technologies, CA, USA) and Bioanalyzer 2100 system (Agilent Technologies, CA, USA), respectively. The quality of the RNA samples met the experimental requirements (RIN> = 7 28S/18S> = 0.7) and were used for RNA sequencing.
Ribosomal RNA was removed using the Ribo-Zero Gold rRNA Removal Kit (Illumina), and the remaining RNA was fragmented by a fragmentation reagent. Then, the libraries were constructed using TruSeq Stranded Total RNA with Ribo-Zero Gold (Illumina, Cat. no. RS-122-2301) according to the manufacturer’s instructions. Whole transcriptome sequencing, including circRNA sequencing and mRNA sequencing, was carried out on an Illumina HiSeq 2500 (OE Biotech, Shanghai, China), and the reading length was 2 × 150 BP (pe150).