Peter van Mourik

31 R117H function and VX-770 response in organoids instructions. Total RNA (500 ng) of each organoid sample was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Inc.) following the manufacturer’s instructions. RT-qPCR reactions to determine R117H-CFTR expression were performed on a Bio-Rad CFX96 Real Time Detection System using a two-step RT-qPCR protocol and SYBR Green Supermix (BioRad). Each experiment included standard curves to determine reaction efficiency, together with melting curve analyses to verify amplification specificity and absence of primer dimers. Gene expression was quantified using CFX Manager™ Software (Bio-Rad) qPCR analysis software. Raw CFTR cycle of quantification (Cq) values were normalized using ACTB and YWHAZ housekeeping genes and converted into relative quantities using the R117H-homozygous organoid cell line as reference sample. Three technical replicates per patient were included in each experiment (n=3). Reference gene selection and validation To select the most stable reference genes for our RT-qPCR experiments, ten commonly used reference genes were tested based on existing literature 4 : beta- actin ( ACTB ), glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ), beta-2 microglobulin ( B2M ), glucuronidase beta ( GUSB ), hydroxymethylbilane synthase ( HMBS ), hypoxanthine phosphoribosyltransferase ( HPRT1 ), TATA-box binding protein ( TBP ), succinate dehydrogenase complex, subunit A, flavoprotein ( SDHA ), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta ( YWHAZ ) and ribosomal protein S13 ( RPS13 ). Primer sequences and amplicon sizes are shown in Supplementary table 2. RT-qPCR was performed as described above. Individual cDNA samples of eight of the 14 intestinal organoids were used. Reference gene stability and the optimal number of reference genes were evaluated using the web-based tool RefFinder (https://www.heartcure.com.au/reffinder/ ) and the GeNorm algorithm 5 . The optimal number of reference genes for our experiments was two (GeNorm V coefficient <0.15). The three most stably expressed reference genes in the organoid samples were B2M, YWHAZ and ACTB according to GeNorm (GeNorm M coefficients <0.5) and ACTB, YWHAZ and GUSB according to RefFinder (data not shown). As such, ACTB and YWHAZ were included as reference genes in our RT-qPCR experiments. R117H-CFTR allele-specific primer design and validation In supplementary table 2 the (allele-specific) primer sequences are depicted that were used in the R117H-CFTR gene expression studies. A forward primer spanning the exon-exon junction of exons 3 and 4 was designed, together with reverse primers targeting the wild type CFTR sequence or the R117H-CFTR sequence (G→A). In the R117H-CFTR allele-specific reverse primer a deliberate mismatch was introduced 2