Single-cell Transcriptomics of Organoid Cultures

At d100, cell lines used were 1013 and 1581 for WT and L75Pfs clones 9 and 11. D170 organoids were 1013 for WT and clones 7 and 9 for L75Pfs. Organoids were dissociated to single cells with papain (Worthington) to 1 mg/ml and 5 μL DNase (Roche) per mL, using 200 uL papain mix per organoid. After 1–2 h when organoids appeared fully dissociated the reaction was quenched with media containing 10% FBS (Gibco). Single cells were resuspended in HBSS (Gibco) and 0.1 mg/mL BSA at 120,000 cells per mL. Single-cell capture was performed using a home-made Dropseq setup according to the published Dropseq protocol^{21 (link)}. Cells were combined in oil (Biorad) and barcoded beads (Chemgenes) in ~1 nL droplets. Droplets were broken using 6× SSC and perfluorooctanol (Sigma) to collect beads. Reverse transcription was performed using Maxima reverse transcriptase (Thermo Fisher Scientific) and cDNA was amplified using Kapa (Roche). cDNA was quantified using a Bioanalyzer DNA High Sensitivity Chip (Agilent). cDNA was fragmented and libraries were created using the Nextera XT library prep kit (Illumina). Libraries with quantified by Qubit dsDNA HS (Thermo Fisher Scientific) and sequenced via Illumina HiSeq 2500.