GSM7508528: HiC RPE/choroid, replicate 3; Homo sapiens; Hi-C
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 nuclei were aliquoted per 10 million and snap frozen after supernatant removal. Samples were stored at -80°C. Crosslinked nuclei from four neural retina and four RPE/choroid samples were used to construct Hi-C libraries, following the in situ Hi-C protocol adopted by the 4D Nucleome consortium with a few adaptations. Briefly, for each replicate ~5 million pre-lysed, crosslinked nuclei were digested overnight using 250 U DpnII restriction enzyme (New England Biolabs, R0543L). DNA ends were marked by incorporating biotin-14-dATP (Life Technologies, 19524-016) and ligated for 4 hours using 2000 U T4 DNA ligase (New England Biolabs, M0202L). Subsequently, crosslinks were reversed overnight using proteinase K (Qiagen, 19131) and Hi-C template DNA was purified using 1x AMPure XP beads (Beckman Coulter, A63881) and stored at 4°C until library preparation. Hi-C template DNA was sheared to a size of 300-500 bp using microTUBE snap-caps (Covaris, 520045) in a Covaris M220 sonicator and MyOne Streptavidin T1 beads (Life Technologies, 65601) were used to pull down biotinylated ligation junctions. Next, samples were split into 5 µg aliquots for sequencing library preparation using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, E7645L) and NEBNext Multiplex Oligos (New England Biolabs, E7335L). Amplified libraries were purified and size selected using 0.55x and 1.2x AMPure XP beads (Beckman Coulter, A63881). Pooled libraries were sequenced on an Illumina NovaSeq 6000 using 100 bp paired-end reads to a depth of ~500 million reads per sample. in situ Hi-C