Total RNA isolation, library preparation, and sequencing

Total RNA was isolated from each sample (63 tumor samples and 14 paired adjacent normal samples) using the Qiagen RNeasy formalin-fixed paraffin-embedded (FFPE) Kit (73504, Qiagen, Hilden, Germany), following the protocol from the manufacturer. The purity and quantity of total RNA were measured by Nanodrop. The integrity of RNA was evaluated using the RNA Nano6000 Assay Kit on the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). 1 μg RNA of per sample was used as input for the RNA sample preparations. Strand-specific RNA sequencing libraries were generated using the Whole RNA-seq Lib Prep kit for Illumina (RK20303, ABclonal, Shanghai, China). Library quality was evaluated on the Agilent Bioanalyzer 2100 system (Agilent, USA). Final libraries were sequenced at the Novogene Bioinformatics Institute (Beijing, China) on an Illumina Hiseq X10 platform by 150 bp paired-end reads.