Fastqc software

The sequencing was performed in collaboration with the Genomics Core of the UZ Leuven (Belgium). Samples (n = 3 per group, 2 μg RNA) were prepared by TruSeq library preparation (RS122, Illumina, San Diego, CA, USA) to generate single end unstranded sequencing libraries, according to the manufacturer’s recommendations. Sequencing of all samples was carried out in 1 lane of 1 flow cell on the HiSeq2000 (Illumina), using single end chemistry with read lengths of 50 base pairs. Between 17.3 and 26.5 million reads were sequenced for each sample. The quality of the reads was checked using the FASTQC software (Illumina). All the reads passed the Phred score (≥28).

The quality of raw reads from the Illumina sequencing was analyzed by FastQC software² and filtered as follows: erroneous k-mers were eliminated with r-Corrector software (Song and Florea, 2015 (link)), adaptors and bases with a Phred score lower than 30 were trimmed with TrimmGalore.³ Additionally, reads were mapped against SILVA (LSU/SSU) Database⁴ using Bowtie2⁵ to eliminate rRNA contaminant reads. The filtered reads were de novo assembled using Trinity software⁶ with default parameters, using a de Bruijn graph algorithm and a k-mer length of 25. The quality of transcriptome assembly was evaluated by mapping the reads over the assembled contigs with bowtie2. Completeness was analyzed with Benchmarking Universal Single-Copy Orthologs (BUSCO) using the Arthropoda odb9 database,⁷ and the number of fully reconstructed coding transcripts was evaluated by a BLASTx search against Swiss-Prot database with a cut off E-value ≤ 1e-20. All raw reads have been deposited in the sequence reads archive (SRA) at National Center for Biotechnology Information (NCBI), and may be accessed using the access code PRJNA772728.

The blood collection procedure used a needle for blood collection into anticoagulant tubes containing either ethylenediaminetetraacetic acid (EDTA) or heparin. After blood collection, the plasma was separated and frozen until further analysis. RNA was extracted from spleen tissues by employing Trizol reagent (Invitrogen, Waltham, MA, USA). After quality assessment, RNA samples were sequenced by constructing libraries on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA). Quality control was performed on the raw Illumina sequences using FASTQC software (version 0.11.7) to obtain clean reads for subsequent analysis. The paired-end reads obtained by Illumina sequencing were compared with the reference genome (Sus scrofa 11.1) using Hisat2 software (version 2.0.5).

RNA was extracted from KYSE150 and KYSE180 cells using TRIzol (Beyotime, China). The extracted RNA samples were first examined for concentration and purity to exclude degradation or contamination using a Qubit and Nanodrop spectrophotometer. For library construction, nonstranded cDNA libraries were constructed using poly(A) mRNA enrichment and the NEBNext UltraTM II RNA Library Prep Kit for Illumina following the manufacturer’s instructions. RNA and library qualities were confirmed using fragment analysis (Agilent 2100 Bioanalyzer). FastQC software (version 0.11.7) was used for quality control of the raw data, and Illumina was used to evaluate the sequencing error rate and base quality. For gene differential expression analysis, EBSeq was used to obtain the differentially expressed gene sets between the two samples, and fold change ≥ 2 or ≤ 1/2 and FDR < 0.01 were used as the screening standards. To investigate differences in the biological processes associated with these DEGs, Gene Ontology (GO) enrichment was performed (Q ≤ 0.05 was considered to indicate significant enrichment). Next, the enriched annotated genes were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analysis.

RNA sequencing was carried out by Nestlé Research (Lausanne, Switzerland). The cDNA libraries were generated using the TruSeq Stranded mRNA Kit (Illumina), followed by PCR amplification for sequencing on Illumina HiSeq 2500. Paired-end cDNA libraries were generated from all samples, and sequencing was performed to generate the ~ 125 bp paired-end reads. FastQC software (v0.11.5) was used for quality control, and assessment of raw Illumina reads in FASTQ format to obtain per base quality, guanine-cytosine (GC) content, and sequence length distribution. Low-quality reads, adapters, and poly-N-containing reads were removed from the raw data. Approximately 95% of high quality reads were obtained in each library from generated data. An average of 90 million paired-end reads was obtained for each library.