Total RNA was extracted from tumor and normal urothelial tissues using TRIzol (Invitrogen, CA, USA) according to the manufacturer's instructions. RNA sequencing libraries were prepared from >500 ng of total RNA using the NEBNext® UltraTM RNA Library Prep Kit (NEB, USA) according to the manufacturer's instructions. Briefly, poly(A)+ mRNA was enriched using Oligo(dT)25 magnetic beads and fragmented in fragmentation buffer. First-strand cDNA was synthesized using ProtoScript® II Reverse Transcriptase with random primers, followed by second-strand cDNA synthesis using DNA polymerase I. The double-stranded cDNA was then end-repaired, a-tailed, and ligated with sequencing adapters. Fragments of approximately 250 bp were selected using magnetic beads, followed by PCR amplification and purification to generate the final sequencing libraries.