R-C2
Total RNA was isolated from liver metastasis samples using Trizol Reagent (Invitrogen, USA), followed by genomic DNA removal with DNase I (TaKaRa, Shiga, Japan). After assessing RNA concentration, purity, and integrity, total RNA samples with a minimum quantity of 1 g were selected for library preparation. Polyadenylated mRNA was enriched using Oligo(dT) magnetic beads and subsequently fragmented randomly via ion-mediated cleavage in the presence of divalent cations, employing the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs Inc; Ipswich, Massachusetts, USA). First-strand cDNA synthesis was performed using fragmented mRNA as template and random oligonucleotides as primers. The double-stranded cDNA was purified, followed by end repair, adenylation of 3' ends, and adapter ligation. cDNA fragments ranging from 400 to 500 bp were size-selected using AMPure XP beads, amplified by PCR, and further purified with AMPure XP beads to construct the final library. Library quality was assessed using the Agilent 2100 Bioanalyzer with the Agilent High Sensitivity DNA Kit (Agilent Technologies Inc, California, USA, 5067-4626). The total library concentration was quantified via PicoGreen fluorescence measurement (Quantifluor-ST fluorometer, Promega, Madison, Wisconsin, USA, E6090; Quant-iT PicoGreen dsDNA Assay Kit, Invitrogen, California, USA, P7589), and the effective library concentration was determined by quantitative PCR (StepOnePlus Real-Time PCR Systems, Thermo Scientific, Waltham, Massachusetts, USA). Multiplexed DNA libraries were normalized and pooled in equal volumes. The pooled library was subjected to stepwise dilution and quantification prior to sequencing on an Illumina platform under PE150 mode.