Protocol for generating and characterizing Matrigel-free prostate cancer patient-derived organoids

Here, we present a protocol for the generation of prostate cancer patient-derived organoids (PCa PDOs) in Matrigel-free conditions. We describe steps for sample selection, pathological resection, and tissue dissociation. We then detail procedures for the generation, maintenance, and characterization of the PCa PDOs. Finally, we provide guidance for the processing of PDOs for single-cell RNA sequencing (scRNA-seq) analysis and using the prostate PDO single-cell atlas (PPScA) for both novice and advanced bioinformatic users. For complete details on the use and execution of this protocol, please refer to Dolgos et al.¹.