EXPERIMENT_TITLE: GSM6431766: PGT_HiC_Rep2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: For in-situ Hi-C, H9 cells and their in vitro differentiated organoids were collected, fixed with 1% formaldehyde for 10 min at room temperature and quenched with glycine, washed with PBS and stored frozen at -80°C. For library preparation, fixed organoids were gently homogenized in Hi-C lysis buffer with pestle A to release nuclei. To derive the proximity ligation products of chromatin, DpnII digestion, biotin-dCTP fill in and re-ligation were applied to released nuclei sequentially. To extract and purify re-ligated DNA, samples were digested by Proteinase K, reverse-crosslinked at 65°C overnight, and then precipitated by ethanol to get pure DNA. Purified DNA were further sheared by sonication to 200-500 bp in length. After that, streptavidin dynal beads were incubated with sheared DNA to pull down biotin-labeled proximity ligation products for library preparation on beads. After end-repair, A-tailing and Truseq adapter ligation, PCR amplification was finally performed by using Truseq barcoded primers to generate libraries for Illumina sequencing. Truseq libraries were constructed using the standard protocol. Genomic fragments were end repaired (NEBNext End Repair Module), A-tailed by adding adenosine to the 3' ends of fragment using Klenow fragment (3' to 5' exo minus, New England Biolabs), and adaptors were ligated at room temperature for 1 hr with T4 DNA ligase (New England Biolabs). Libraries were amplified with Illumina primers using the KAPA SYBR FAST qPCR Master Mix.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Transcriptional reprogramming during cell fate determination involves precise targeting of enhancers to tissue-specific genes. Enhancer-promoter interactions are regulated by different types of 3D chromatin organization, including compartmental domains and CTCF loops. To understand the contribution of these different levels of chromatin organization in cell lineage commitment, we analyzed changes in 3D organization during the differentiation of human embryonic stem cells (hESCs) into pancreatic islet organoids. We find that major events in chromatin reorganization, including decompaction of transposon-enriched heterochromatin, breakdown of poised enhancer-promoter interaction networks, gain or loss of CTCF loops and establishment of novel enhancer-promoter contacts, occur either sequentially or simultaneously. Transcriptionally activated transposons released from unstable heterochromatin compartments recruit CTCF to form new loops. In contrast, when released from poised long-range interaction networks, enhancers and genes crucial for cell differentiation recruit tissue-specific transcription factors by either direct binding or indirect 3D contacts containing RNA Polymerase II. Activation of stage-specific genes correlates with gain of CTCF binding at anchors of extended CTCF loops, suggesting changes in loop extrusion capacity. In agreement with this, we find a global increase in cohesin loading leading to longer residency on extended CTCF loops encompassing tissue-specific genes. Our findings provide new insights into how distinct types of chromatin organization affect the reprogramming of gene expression patterns to regulate cell lineage commitment. Overall design: Hi-C
SAMPLE_TITLE: PGT_HiC_Rep2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "H9 hESCs"}, {"TAG": "developmental stage", "VALUE": "PGT stage"}, {"TAG": "cell line", "VALUE": "H9 hESCs"}, {"TAG": "cell type", "VALUE": "in vitro differentiated PGT cells"}, {"TAG": "genotype", "VALUE": "WT"}, {"TAG": "treatment", "VALUE": "Pancreatic differentiation"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: PGT_HiC_Rep2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: PGT_HiC_Rep2
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR20825721.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos2/sra-pub-zq-18/SRR020/20825/SRR20825721/SRR20825721.lite.1", "size": "27163075341", "date": "2022-11-16 20:56:30", "md5": "f0aaf31887f648ca3e82a6b8849fc2f9", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR20825721", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR20825721/SRR20825721", "size": "32721440089", "date": "2022-08-04 21:28:33", "md5": "dbc0fc8524c7cd44495d010c3157c049", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "PGT_DpnII_HiC_Rep2_R2.fastq.gz", "size": "31467100529", "date": "2022-08-04 21:23:01", "md5": "c28bb48c519b351fb00b3e9ed17e85f7", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "PGT_DpnII_HiC_Rep2_R1.fastq.gz", "size": "31075456478", "date": "2022-08-04 21:20:07", "md5": "96e90d470231a1685ffc2c44db03359d", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}]