title: Canis lupus familiaris HCC, HCA RNA-seq
design: RNA extraction from canine tissues: Total RNA was isolated from canine liver tissues using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific Inc.) in accordance with the manufacturers protocol, and the RNA concentration of each sample was subsequently quantified using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific Inc.), while RNA quality and integrity were assessed via electrophoretic analysis on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The average RNA Integrity Number (RIN) across all samples was 8.5, with individual values ranging from 8.0 to 9.6, and that electropherograms consistently showed a distinct peak within the small RNA region (~25 nucleotides), thereby confirming the suitability of the extracted RNA for downstream small RNA sequencing applications. Construction and sequencing of small RNA libraries: Small RNA libraries for Health 13 and HCC 13 samples were prepared and sequenced by Hokkaido System Science Co., Ltd. (Sapporo, Japan) using the TruSeq Small RNA Sample Preparation Kit (Catalog # RS-200-0012, Illumina Inc., San Diego, CA, USA), following the manufacturer's protocol without deviation, and were subsequently sequenced on the Illumina HiSeq 2500 platform (Illumina Inc., San Diego, CA, USA) using single-end mode with a read length of 50 base pairs. In contrast, small RNA libraries for HCA 15 samples were processed by Azenta Life Sciences (Chelmsford, MA, USA) using the QIAseq miRNA Library Kit (QIAGEN GmbH, Hilden, Germany), also following the manufacturers instructions. These libraries were sequenced on the Illumina NovaSeq 6000 platform (Illumina Inc., San Diego, CA, USA) using single-end mode with a read length of 75 base pairs. Product numbers for the QIAseq kits were not disclosed by the service provider.