Suzanne de Bruijn

192 Chapter 4 melting curve for each reaction. The fold difference of the target region was normalized to the wildtype reference genomic region with respect to the calibrator sample, and was calculated using the ΔΔCt method. 15 Interrogation of the genomic region Available Hi-C, ChIP-seq and RNA-seq datasets were downloaded, analyzed and visualized using UCSC genome browser. 16 Human retina ChIP-seq and RNA-seq datasets were obtained from Cherry et al. 2020. 17 CTCF ChIP-seq datasets for GM12878 and K562 were retrieved from the ENCODE project/Broad Institute 18 and for MCF-7 from the ENCODE project/University of Washington. 19 CTCF ChiA-PET libraries for K562 and MCF- 7 (GSM970215) were obtained from the ENCODE/GIS-Ruan dataset. 20 Reprogramming fibroblasts into iPSCs and differentiation into photoreceptor progenitor cells and 3D retinal organoids Fibroblasts were cultured from skin biopsies of individuals with NL-SV1, UK-SV2, and anonymous control individuals. Cell lines were reprogrammed into iPSCs and differentiated in PPCs (NL-SV1) or ROs (UK-SV2). For NL-SV1, fibroblasts of two affected and four anonymous control individuals were reprogrammed into iPSCs. Reprogramming into iPSCs was performed by lentiviral transduction as previously described 21 , for one control cell line, reprogramming was performed using episomal vectors (Addgene). 22 iPSC lines for each affected and control individual were then differentiated into PPCs following the previously described 60-day protocol. 21,23 For each iPSC line, differentiation was performed for two iPSC clonal lines in triplicate. Differentiation of PPCs was confirmed by RT-qPCR for neural ( PAX6 ) and photoreceptor progenitor ( CRX and NRL ) markers (data not shown). For one affected individual with UK-SV2 and one control individual, fibroblasts were reprogrammed into iPSCs using episomal vectors (Addgene), as described previously. 22 Retinal organoids were differentiated from iPSC, following a previously described protocol with slight modifications. 24 iPSCs were seeded on plates coated with Geltrex (ThermoFisher Scientific) until neuronal retinal vesicles (NRVs) appeared. NRVs were excised by a sterile scalpel and distributed in single wells in 25 wells low-attachment plates. NRVs were than cultured in Retinal differentiation media; 3:1 v/v of DMEM:F12, 2% B27 supplement, 1% Non-Essential Amino Acid, 1% Penicillin-Streptomycin (Gibco) for one week. Optic vesicles were then cultured in Retinal Maturation Medium 1 (3:1 v/v of DMEM:F12, 2% B27 supplement, 1% Non-Essential Amino Acid, 1% Penicillin- Streptomycin, 10% Fetal Bovine Serum (Labtech), 100 µM Taurine, 2 mM GlutaMAX) until day 70, then changed to Retinal Maturation Medium 2 (3:1 v/v of DMEM:F12,