EXPERIMENT_TITLE: GSM7508528: HiC RPE/choroid, replicate 3; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: 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
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Vision depends on the functional interplay between the photoreceptor cells of the neural retina and the supporting cells of the underlying retinal pigment epithelium (RPE). Many genes involved in inherited retinal diseases (IRD) display highly specific spatiotemporal expression within these interconnected retinal components through the local recruitment of cis-regulatory elements (CREs) in 3D nuclear space. To understand the role of differential chromatin architecture in establishing tissue-specific expression patterns at IRD loci in the human neural retina and the RPE, we mapped genome-wide chromatin interactions by applying in situ Hi-C and H3K4me3 HiChIP to human adult post-mortem donor retinas. A comparative 3D genome analysis between neural retina and RPE revealed that almost 60% of known IRD genes were marked by differential cis-regulatory interactions or 3D genome structure. Furthermore, we zoomed in on tissue-specific chromatin interactions at the ABCA4 locus, which is implicated in the most common autosomal recessive IRD. We constructed high-resolution ABCA4 interaction profiles using UMI-4C, which, upon integration with bulk and single-cell epigenomic datasets and in vivo enhancer assays in zebrafish, revealed tissue-specific CREs for ABCA4. In summary, through extensive comparative 3D genome mapping, based on genome-wide (Hi-C), promoter-centric (HiChIP) and locus-specific (UMI-4C) assays of human neural retina and RPE, we have shown that gene regulation at key IRD loci is likely mediated by tissue-specific chromatin interactions. These findings do not only provide insight into tissue-specific regulatory landscapes at retinal disease loci, but also delineate the search space for genomic variation underlying unsolved IRD. Overall design: in situ Hi-C on RPE/choroid and neural retina from human adult post-mortem donor samples
SAMPLE_TITLE: HiC RPE/choroid, replicate 3
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "RPE/choroid"}, {"TAG": "tissue", "VALUE": "RPE/choroid"}, {"TAG": "developmental stage", "VALUE": "Adult"}, {"TAG": "genotype", "VALUE": "wild-type"}, {"TAG": "treatment", "VALUE": "untreated"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: HiC RPE/choroid, replicate 3
RUN_SET_RUN_Pool_Member_@attributes_sample_title: HiC RPE/choroid, replicate 3
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": null, "@attributes": {"cluster": "public", "filename": "HiC_RPE_rep3_1.fastq.gz", "size": "25633072389", "date": "2023-06-26 09:42:56", "md5": "1cd70925effb1bef5f5a77028a4af425", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "HiC_RPE_rep3_2.fastq.gz", "size": "26544452109", "date": "2023-06-26 09:41:28", "md5": "8dae49f6ae104aef4f620b97cf3ef786", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR25020643", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR25020643/SRR25020643", "size": "32457758503", "date": "2023-06-26 09:45:37", "md5": "45db48ac98ee5a2c581fbd25ead40bdd", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR25020643.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos6/sra-pub-zq-40/SRR025/25020/SRR25020643/SRR25020643.lite.1", "size": "24959008919", "date": "2023-07-01 10:53:12", "md5": "1eae77be2b4f73f0fa4ec8d6c1613e0a", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}]