Qubit 2

Total RNA was prepared with QIAzol Lysis Reagent and RNeasy MinElute Cleanup Kit (Qiagen, Germany). The RNA quality and integrity were analyzed by Qubit 2.0(Life Technologies, USA) and Bioanalyzer 2100 (Agilent, Germany). For library preparation, 3 μg total RNA were captured by NEBNext Oligo d(T) 25 beads (NEB, USA), sheared to fragments of ~ 250 bp, and reverse transcripted by NEBNext RNA first and second Strand Synthesis Module (NEB, USA). The products were end-repaired, A-tailed, and ligated to Illumina sequencing adapters and amplified by PCR. The sequencing library were qualified by Qubit 2.0 (Life technologies, USA) and Bioanalyzer 2100 (Agilent, Germany), then sequenced on Illumina Hiseq X-Ten with 2 × 150 bp paired-end sequencing, which were controlled by Hiseq Control Software (HCS).

Peripheral venous blood samples was subjected to density centrifugation using Ficoll-hypaque solution to isolate mononuclear cells and granulocytes. After lysing RBCs, cells were incubated with BB515 Rat anti-CD11b (BD Biosciences) and BV650 mouse anti-human CD16 (BD Biosciences) antibodies and sorted by flow cytometry using BD FACS AriaII. RNA sequencing (RNA-seq) was performed on sorted CD11b⁺CD16⁺myeloid cells in capecitabine-sensitive patients and CD11b⁺CD16⁻myeloid cells in capecitabine-resistant patients. Qubit 2.0 (Life Technologies, USA) and Bioanalyzer 2100 (Agilent, Germany) were used to analyze the RNA quality and integrity. A total of 3 μg RNA was used for the RNA sample preparations. Extracted RNA samples were processed using the NEBNext® UltraTM RNA Library Prep Kit (Lexogen) and sequenced on an Illumina Hiseq X-Ten with control of Hiseq Control Software (HCS). The sequencing library were qualified by Qubit 2.0 (Life technologies, USA) and Bioanalyzer 2100 (Agilent, Germany). Raw reads were processed through in-house perlscripts. Clean reads were obtained by removing reads containing adapter. Differentially expressed genes were defined by P < 0.05 and an absolute fold change > 2. Using Gene Set Enrichment Analysis, enrichment of a specific gene set was tested, and core enrichment genes were determined.

The RNA quantity and integrity were measured with Qubit 2.0 (Life Technologies, ThermoFisher Scientific, Waltham, MA, USA) and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively. A small RNA library was prepared using a Next Ultra small RNA Sample Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). The total RNA was ligated with a 5′ and 3′ adapter sequentially and then reversely transcribed into cDNA. After cDNA PCR amplification and gel purification, the insert size and quantity of the constructed library were assessed with a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and Qubit 2.0 (Life Technologies, ThermoFisher Scientific, Waltham, MA, USA), respectively. The sequencing procedure was performed with an Illumina HiSeq Xten system at BioMarker Technologies in Beijing, China. The total sRNA reads, including miRNAs and unannotated RNAs, were then obtained by removing sequences from other non-coding RNAs, such as rRNA, tRNA, snRNA and snoRNA in a repetitive sequence using the bowtie tool to align against the Silva, GtRNAdb, Rfam and Repbase databases.

The RNA was isolated using the mirVana kit following the manufacturer’s instructions. Briefly, the cell cultures were washed thrice with PBS to eliminate cellular debris and medium culture traces. The cells were treated with the disruption solution from the kit and then transferred to the affinity columns provided by the manufacturer to eliminate contaminants and finally elute the purified RNA in a clean tube. The purified RNA was quantified using a fluorometric assay in a Qubit 2.0 following the manufacturer’s instructions with the RNA BR assay kit (Thermofisher).
For sequencing, we used a Custom AnyDeplete kit (Tecan), which we designed to carry out the depletion of Atlantic salmon rRNA (Supplementary File 1). Library preparation was performed with Universal plus RNA-seq library preparation kit with NuQuant (Tecan) following the instructions provided by the manufacturer. Library quantity and quality were assessed by Qubit 2.0 (Thermofisher) and TapeStation D1000 ScreenTape (Agilent Technologies Inc.), respectively. The libraries obtained have 350 bp with an insert of 200 bp, including the Illumina 8−nt unique dual indices. Sequencing was performed in an Illumina NovaSeq S4 device, with a configuration of 150 pair-end for 50 million pair-end reads per sample.