GSM7508577: UMI-4C in human RPE/choroid, ABCA4 promoter as viewpoint, RPE_3_L_ABCA4_UMI_1; Homo sapiens; OTHER
Post-mortem human neural retina and RPE/choroid mixtures were obtained through the Tissue Bank of Ghent University Hospital and Antwerp University Hospital under ethical approval of the Ethics Committee of Ghent University (2018/1072, B670201837286). Eye globes were provided with a description of time and cause of death, post-mortem circulation time, age and sex. None of the 6 donors had a prior known ophthalmological condition.  The eye globes were dissected on ice, followed by extraction of the neural retina and the retinal pigment epithelium/choroid. The resulting tissues were resuspended in 1XPBS supplemented with 10% Fetal Bovine Serum. The samples were processed according to Matelot & Noordermeer (2016) and cross-linking of nuclei was performed using 2% formaldehyde. Finally, the obtained nucleai were aliquoted per 10 million and snap frozen after supernatant removal. Samples were stored at -80°C. The generation of the 3C template was performed according to Schwartzman et al. 2016. Briefly, around 5 million cross-linked nuclei were digested overnight using 400 U DpnII (NEB). After digestion, ligation was performed overnight using 4,000 U of T4 DNA ligase (NEB), followed by the addition of proteinase K (BIOzymTC). Efficiency of digestion and ligation were evaluated via agarose gel electrophoresis. Next, samples were de-crosslinked, followed by purification of samples using AMPure XP beads (Agencourt). Subsequently, 4 µg of the 3C template was sheared on a Covaris M220 focused ultrasonicator to get 300 bp DNA fragments. The UMI-4C sequencing library preparation was obtained using the NEBNext Ultra II Library Prep Kit (NEB). Library amplification was performed by nested PCR. In the first PCR, 100ng of library were amplified using an upstream (US) forward primer and a universal reverse primer using the KAPA2G Robust HotStart ReadyMix (Roche). The resulting product was amplified using a downstream (DS) forward primer and the same universal reverse primer. Preparation of libraries was performed as described for UMI-4C libraries assays in Schwartzman et al., 2016, using the NEBNext Ultra II Library Prep Kit (NEB)