Peter van Mourik

30 Chapter 2 SUPPLEMENTARY DATA Supplementary table 1. Genotypes of intestinal organoids used in this study. Organoid number Mutation 1 Mutation 2 Poly-T-tract 1 p.Arg117His p.Arg117His 7T 2 p.Arg117His p.Phe508del 7T-9T 3 p.Arg117His p.Phe508del 7T-9T 4 p.Arg117His p.Phe508del Unknown 5 p.Arg117His p.Phe508del 7T-9T 6 p.Arg117His p.Phe508del 7T-9T 7 p.Arg117His p.Phe508del 7T-9T 8 p.Arg117His p.Arg553X 7T-9T 9 p.Arg117His p.Arg1162X 7T-9T 10 p.Arg117His c.1857delT 7T-9T 11 p.Arg117His p.Phe508del 7T-9T 12 p.Arg117His p.Phe508del 7T-9T 13 p.Arg117His p.Phe508del 7T-9T 14 p.Arg117His p.Trp1282X 7T-9T Forskolin-induced swelling (FIS) assay FIS was performed as described previously 1–3 . In short, organoids were seeded in a 96-well tissue culture-treated plate in 4 μl 50% Matrigel® droplets immersed in 50 μL culture medium. Following overnight incubation at 37°C/5% CO 2 organoid cultures were incubated with 750 nM calcein green (Life Technologies: Gibco) for 30 minutes at 37°C/5% CO 2 . Forskolin (0.050 μM or 0.128 μM) (Sigma), together with 3 μM VX-770 (Selleck Chemicals LLC) or carrier (DMSO), was added directly to the organoids followed by analysis using live-cell confocal microscopy (Zeiss LSM 800) for 60 minutes at 37°C/5% CO 2 . Four technical replicates per patient were included in each experiment (n = 3). The total increase in organoid area relative to t=0 min was calculated using Zen Blue 2.0 analysis software (Zeiss) and results are expressed as area under the curve (AUC) at t=60 minutes. RNA isolation and gene expression studies Primer sequences and amplicon sizes are depicted in Supplementary table 2. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Inc), including DNase I treatment of the samples (RNAse-Free DNase Set, Qiagen), according to the manufacturer’s instructions. The purity of RNA was assessed using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) and quantity was measured using the Qubit Fluorometer (Thermo Fisher Scientific) according to the manufacturer’s