Organoid Establishment, Sequencing and Culture

Colorectal cancer organoids were established from fresh colorectal cancer tissues (Human Tissue Act License number 12121, REC 12-EE-0493 and 18-EE-0025). The establishment and propagation of the organoids was based on previously published protocols (17). Somatic mutations for the p53 mutant sample were determined by the South London Medicine Centre and genomic DNA for the p53 WT organoid was sequenced by the Advanced Sequencing Facility at the Francis Crick Institute.

Colorectal organoids (see Table S2) were seeded by resuspension in basement membrane extract (BME) with media (70/30 Ratio). Plates were left for 30 minutes at 37°C and 5% CO₂ for BME to solidify before the addition of media supplemented with of 10 μM Rock inhibitor Y-27632. For passaging, organoids were dissociated by resuspension in TrypLE Express for 15 minutes at 37°C. Dissociation was stopped by addition of 5% FBS, organoids were further disrupted by pipetting multiple times and then filtered through a 70 μM cell strainer before replating.