library_construction_protocol: RNAs were extracted from pancreatic tumors established from control (wildtype) KPC1361 or Pikfyve-null KPC1361 in C57BL/6 mice. Tumor tissues were lysed with a tissuelyser in QIAzol lysis reagent, and RNAs were extracted with the RNeasy Mini kit (Qiagen), according to the instructions from the manufacturer. The Eukaryote Total RNA Nano kit (Agilent Technologies; catalog #: 5067-1511) was used to check the quality of RNAs on an Agilent bioanalyzer. A total of 800ng RNA was then used for library preparation with the KAPA RNA Hyper+RiboErase HMR kit (Roche Diagnostics; Catalog #: 08098140702), following the instructions from the user manual. Briefly, the ribosomal RNA was removed by enzymatic digestion, and then the RNA was fragmented to around 200-300 bp with heat in fragmentation buffer. Synthesis of cDNA was conducted with reverse transcriptase and random primer, and then the second strand was synthesized to generate double-stranded cDNA. Following the repair of the DNA ends, NEB adapter was ligated, and the DNA are amplified with the KAPA HiFi HotStart mix and NEB dual barcode. Quality of the library was checked using the bioanalyzer with the Agilent DNA 1000 Kit (Agilent Technologies; catalog #: 5067-1504), and the sequencing was performed using NovaSeq 6000.