EXPERIMENT_TITLE: GSM7102115: B14804_KO_neuron_HiC_rep2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: We used the Arima HiC kit to process the samples. Briefly, frozen cell pellets were lysed and conditioned before chromatin digestion. The digested chromatin was then filled in and biotinylated before ligation. Next, chromatin was protein-digested and reverse crosslinking overnight, followed by purification. The purified DNA was then sonicated using the bioruptor pico (Diagenode) and size selected before library preparation using the NEB DNA Ultra II, following Arima's protocol. The Hi-C DNA was bound to streptavidin beads before enzymatic end prep, adaptor ligation, DNA release by heat incubation, and lastly, PCR to barcode and amplify the libraries.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Topological associating domains (TADs) are self-interacting genomic units crucial for shaping gene regulation patterns. Despite their importance, the extent of their evolutionary conservation and its functional implications remain largely unknown. In this study, we generate Hi-C and ChIP-seq data and compare TAD organization across four primate and four rodent species, and characterize the genetic and epigenetic properties of TAD boundaries in correspondence to their evolutionary conservation. We find 14% of all human TAD boundaries to be shared among all eight species (ultraconserved), while 15% are human-specific. Ultraconserved TAD boundaries have stronger insulation strength, CTCF binding, and enrichment of older retrotransposons, compared to species-specific boundaries. CRISPR-Cas9 knockouts of two ultraconserved boundaries in mouse models leads to tissue-specific gene expression changes and morphological phenotypes. Deletion of a human-specific boundary near the autism-related AUTS2 gene results in upregulation of this gene in neurons. Overall, our study provides pertinent TAD boundary evolutionary conservation annotations, and showcase the functional importance of TAD evolution. Overall design: We sequenced H3K4me3, H3K27me3, H3K4me1, H3K27ac, and CTCF ChIP-seq of three rodent species liver tissue, and LCL from two gibbon species. A Male and female biological replicate were sequenced for each mark and species. To test functionality of human-specific boundaries, we used CRISPR to knock-out a human-specific TAD boundary (two replicated) in iPSC-induced neurons, and used HiC to compare chromatin interaction between wild-type (two replicated) and knock-out cells. To test functionality of ultraconserved boundaries, we used CRISPR to knock-out a candidate ultra-conserved TAD boundaries in mouse, and used Capture HiC to compare chromatin interaction in the heart of wild-type (two replicates) and knock-out mice (two replicates).
SAMPLE_TITLE: B14804_KO_neuron_HiC_rep2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "iPSC-derived human neurons"}, {"TAG": "tissue", "VALUE": "iPSC-derived human neurons"}, {"TAG": "cell line", "VALUE": "WTC11 Ngn2 derived"}, {"TAG": "genotype", "VALUE": "homozygous B14804 KO"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: B14804_KO_neuron_HiC_rep2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: B14804_KO_neuron_HiC_rep2
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR23883911", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos4/sra-pub-run-30/SRR023/23883/SRR23883911/SRR23883911.1", "size": "24499840381", "date": "2023-03-17 19:55:56", "md5": "4928ffaaa43db4f70f003416309fb3fa", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "AUTS2_7_R2.fastq.gz", "size": "19927152511", "date": "2023-03-17 19:51:40", "md5": "117dd55b5b8458e4d986a73e506de0d1", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "AUTS2_7_R1.fastq.gz", "size": "17770692967", "date": "2023-03-17 19:50:45", "md5": "86689e2a7210a7ab087796e0acbf94b0", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}]