Protocol for isolation and functional validation of label-retaining quiescent colorectal cancer stem cells from patient-derived organoids for RNA-seq.

Quiescent cancer stem cells (qCSCs) are a major source of posttreatment relapse, but methods to identify molecular targets for qCSC elimination are limited. Here, we present a protocol using the fluorescent dye PKH26 to isolate label-retaining qCSCs from colorectal cancer (CRC) patient-derived organoids (PDOs). We describe processing of organoids to single cells, followed by PKH26 labeling and FACS-based cell isolation. We then detail steps for functional assays and RNA sequencing. This protocol can also be applied to normal tissue-derived organoids. For complete details on the use and execution of this protocol, please refer to Regan et al. (2021).