RNA-seq analysis of ex vivo microtumor models comprising control or Arpc4 knockout (KO) PDAC cells

The loss of Arpc4 resulted in reduced expression of other Arp2/3 complex components, rendering the Arp2/3 complex nonfunctional. The ex vivo microtumor(3D) models were composed of either control or Arpc4 knockout (KO) KP 8025 cells, mouse primary pancreatic stellate cells (PSCs), and bone marrow-derived macrophages. To identify genes affected by Arpc4 knockout in this model, RNA-seq analysis was performed on bulk samples. R254 cell line which was derived from primary cells isolated from genetically engineered p48Cre/+; LSL-KrasG12D/+; p53flox/flox (KPC) mice, and 8025 cell line which was derived from primary cells isolated from genetically engineered p48Cre/+; LSL-KrasG12D/+ (KC) mice were also conducted with RNA seq. Overall design: The 8025 and R254 cell line were established from primary cells isolated from genetically engineered p48Cre/+; LSL-KrasG12D/+ (KC) and p48Cre/+; LSL-KrasG12D/+; p53flox/flox (KPC) mice, and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained at 37°C in a humidified incubator with 5% CO2. The cell line was transfected with the CRISPR/Cas9 p20-ARC Double Nickase Plasmid (Santa Cruz Biotechnology, USA) to target the Arpc4 gene, alongside a negative control plasmid (Santa Cruz Biotechnology, USA). Transfections were carried out using Lipofectamine 3000 (Thermo Fisher, USA) according to the manufacturer's protocol. Forty-eight hours post-transfection, green fluorescent cells were sorted via fluorescence-activated cell sorting (FACS) and selected using puromycin (InvivoGen, Toulouse, France) at concentrations of 0.7 mg/mL for 8025 cells and 1.2 mg/mL for R254 cells. After 5–7 days of puromycin selection, cells were seeded into 96-well plates for clonal expansion. Arpc4 knockout (Arpc4ko) clones were validated via Western blot analysis. Total RNA was extracted using the Monarch Total RNA Miniprep Kit in accordance with the manufacturer's protocol. RNA sequencing was performed at the Core Unit Bioinformatics, Medical Faculty, Ulm University.Ex vivo microtumor models (3D) were constructed using organoids, mouse primary pancreatic stellate cells (PSCs), and bone marrow-derived macrophages embedded in a matrix consisting of 75% Matrigel, 25% Type I collagen, 5 µg/mL fibronectin, and 2.5 µg/mL vitronectin. Total RNA was extracted by adding TRIzol reagent (Thermo Fisher, USA) to the gel after removing the supernatant and dissolving the gel by thorough mixing. Chloroform (Thermo Fisher, USA) was then added, followed by vigorous vortexing for 15 seconds. After incubation at room temperature for 3 minutes, the mixture was centrifuged at 12,000×g for 15 minutes at 4°C. The upper aqueous phase was carefully transferred to a fresh tube, mixed with an equal volume of =95% ethanol, and purified using the Monarch Total RNA Miniprep Kit (New England Biolabs, Germany) according to the manufacturer's protocol. RNA sequencing was performed at the NGS Core Facility, Medical Faculty Mannheim, University of Heidelberg.