title: RNA-Seq of colon tissue: Male with out PNI
design: Total RNA was extracted from CRC tissues and cultured cells using Trizol reagent (TaKaRa, Japan) following the manufacturers protocol. RNA quality was assessed, and mRNA was isolated, fragmented, and converted into cDNA. Libraries were prepared using the TruSeq RNA Sample Prep Kit (Illumina, USA) and sequenced on an Illumina HiSeq platform. Differential gene expression analysis was performed using DESeq2, with criteria of fold change 2 and adjusted P-value <0.05. Bioinformatics analysis focused on circadian rhythm-associated genes, such as RORC and CRY2, to elucidate their roles in CRC progression.