library_construction_protocol: Tumor RNA was extracted from fresh frozen tumor tissue using Trizol. The tissue block was ground with liquid nitrogen, and 50-100 mg was transferred to a 2.0 mL centrifuge tube containing 1 mL of Trizol. Chloroform (1/5 volume) was added, and the mixture was shaken vigorously, incubated at room temperature for 2-3 minutes, and centrifuged at 12,000 rpm at 4°C for 10-15 minutes. The upper aqueous phase was transferred to a new tube, mixed with an equal volume of isopropanol, and incubated at room temperature for 10 minutes. After centrifugation at 12,000 rpm at 4°C for 10 minutes, the supernatant was discarded, and the RNA pellet was washed with 75% ethanol prepared with DEPC water. The pellet was air-dried, dissolved in RNase-free water (DEPC water), and stored at -80°C. Total RNA was used to generate sequencing libraries using the NEB Next Ultra RNA Library Prep Kit for Illumina (Catalog #: E7530L). mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was performed with divalent cations under elevated temperatures. First-strand cDNA synthesis was conducted using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H). Second-strand cDNA synthesis was performed using DNA Polymerase I and RNase H. Overhangs were converted to blunt ends, and NEB Next Adaptor with a hairpin loop structure was ligated to the adenylated 3' ends of DNA fragments. Library fragments were purified to select 370-420 bp cDNA fragments using the AMPure XP system. USER Enzyme was used for size selection, followed by PCR with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. PCR products were purified, and library quality was assessed using the Agilent 5400 system and quantified by QPCR (1.5 nM).