Suzanne de Bruijn

193 Structural variants cause ectopic enhancer-gene contact in retinitis pigmentosa 1% N2 supplement, 1% Non-Essential Amino Acid, 1% Penicillin-Streptomycin, 10% Fetal Bovine Serum (Labtech), 100 µM Taurine, 2 mM GlutaMAX) until maturation and collection of the ROs for experimental procedures. Media was supplemented with 1 µM retinoic acid from day 50 to day 70, then changed to 0.5 µM from day 70 to day 100. After day 100 no further supplement was added to the media. Preparation of low input Hi-C libraries (Low-C) Four UK-SV2 and four control 200-day old ROs were harvested and dissociated to single cells by gentle trituration in 150 µL PBS. Total volume was brought up to 500 µL with PBS before fixation with 2% PFA/PBS for 10 min while tumbling. Next, 100 µL of 1.425 M glycine were added and incubated in rotation for 5 min. To quench the cross-linking reaction, cells were placed on ice for 10 min. Then, cells were centrifuged for 8 min at 500 g and 4˚C, and supernatant was removed. The pellet was resuspended in cold lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1.15 Triton X-100, 5% Protease inhibitor cocktail) and incubated for 15 min on ice. Cells were centrifuged for 5 min at 500 g and 4˚C, and the supernatant was discarded. Finally, lysed cells were washed in 500 µL PBS and centrifuged for 2 min at 500 g and 4˚C. Cells were snap frozen in liquid N2 before restriction enzyme digestion. Next, RO fixed chromatin (2x10 5 cells) from UK-SV2 and controls was digested for 2h at 37 ˚C with a 4bp cutter ( DpnII ; New England Biolabs - NEB). The DNA overhangs generated by the restriction enzyme were marked with biotin-14-dATP (Thermo Fischer Scientific) and the proximity ligation step was performed for 4h at 18˚C using T4 DNA ligase (NEB). Crosslink reversal was performed overnight at 65˚C with vigorous shaking (1,000 rpm). The DNA was precipitated by adding Phenol-Chloroform-Isoamyl alcohol mix (25:24:1) (Merck) and then sheared to fragments of 300-600 bp using Covaris S220 (2 cycles, each 50sec long; 10% duty; 4 intensity; 200 cycles/burst). The biotin-filled DNA fragments were pulled down using Dynabeads MyOne Streptavidin T1 beads (Thermo Fischer Scientific) and the products were prepared for Illumina short-reads sequencing using the NEBNext Ultra DNA Library Prep kit (NEB). Quantitative real time PCR of genes and enhancer RNA within the RP17-locus Expression of genes located in the RP17-locus was assessed using RT-qPCR in human tissues, affected individual and control PPCs and ROs. Commercially available RNA panels were used to determine the expression of GDPD1 and YPEL2 in healthy human adult tissues. RNA isolation and cDNA preparation were performed as previously described. 25 Single cell RNA sequencing data of human 26 and primate retinal cell types 27 was obtained and visualized using the Broad Institute Single Cell Portal.