library_construction_protocol: The study was approved by the Ethics Committee of USTC (Ethics Approval No.: 2024-RE-359). All pancreatic cancer patients who participated in this study at the No. 1 Affliated Hospital of USTC provided consent forms. None of the patients received any neoadjuvant therapy before tissue collection. Fresh tumor specimens and adjacent mucosal specimens were resected intraoperatively and there were no cancer cells remaining at the end of the resected samples as confirmed by the histopathologist. Representative hemoglobin/eosin (H&E)-stained sections of the excised specimens used for scRNA-seq were delineated with the help of a pathologist to delineate the three adjacent subregions (tumor core, tumor margin, and distal paracancerous region), after which each of the three regions was digested into single-cell suspensions and analyzed using droplet-based scRNA-seq. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq 2500 following the manufacturer's protocols. ﻿