Suzanne de Bruijn

171 Structural variants cause ectopic enhancer-gene contact in retinitis pigmentosa Interrogation of the genomic region We interrogated chromatin and genome regulation datasets to explore the epigenomic landscape of the region. Available datasets were obtained and analyzed using UCSC genome browser (details of datasets used are provided in Supplementary Materials and Methods ). Reprogramming fibroblasts into iPSCs and differentiation into photoreceptor progenitor cells and 3D retinal organoids Fibroblasts were cultured from skin biopsies of two individuals with NL-SV1, one individual with UK-SV2, and five anonymous control individuals. Cell lines were reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into photoreceptor progenitor cells (PPCs) following the previously described 60-day protocol ( Supplementary Materials and Methods ). 18,19 3D retinal organoids (ROs) were differentiated for UK-SV2 and controls, as previously described ( Supplementary Materials and Methods ). 20 Preparation of low input Hi-C libraries (Low-C) Hi-C was performed on UK-SV2 and control 3D ROs using a low input protocol (Low-C) with few modifications ( Supplementary Materials and Methods ). 21 Two libraries per sample were sequenced for 200 million fragments in a 100 bp paired-end run on a NovaSeq 6000 (Illumina). Paired-end sequencing data was processed using Juicer 22 and the Hi-C maps were created using a bin size with 10 kb resolution. Further information about the bioinformatics pipeline is detailed in Melo et al., 2020. 23 Expression analysis of genes and enhancer RNA within the RP17-locus Toassess expressionof genes, qPCRwas performed for different human tissues, including retina ( Table S3 , SupplementaryMaterials andMethods ). Single cell RNA sequencing data of human 24 and primate 25 retinal cell types was obtained and visualized using the Broad Institute Single Cell Portal ( Supplementary Materials and Methods ). cDNA was synthesized from total RNA extracted from PPCs, ROs and fibroblasts. Differential expression of genes implicated in the SVs, and control housekeeping and retinal progenitor genes, were assessed by qPCR ( Table S3 , Supplementary Materials andMethods ). Primersweredesigned to theenhancer regioncontainingmultiple retinal transcription factor binding sites implicated in all SVs, to analyze targeted enhancer RNA expression by qPCR ( Table S3 , Supplementary Materials and Methods ).