EXPERIMENT_TITLE: GSM5024974: ZT-KO2-STAG2-A2-H4-R2-T1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: 10M formaldehyde cross-linked cells were lysed and digested overnight at 37°C with 400 units of DpnII (NEB). Next, the DNA ends were marked with biotin-14-dATP (Thermo Fisher Scientific #19524016) for 4 hours at 23°C and ligated for 4 hours at 16°C. To remove proteins and reverse crosslink, DNA was treated with proteinase K overnight at 65°C. DNA was then purified using phenol:chloroform and biotin was removed from unligated ends. Ligation products were fragmented by sonication to an average size of 200bp and size selected with Ampure XP beads (Beckman Coulter, # A63880) to fragments ranging from 100 to 350 bp. End repair was performed on the DNA fragments to obtain blunt ends and biotinylated ligation products were purified using streptavidin beads (Thermo Fisher Scientific #65001). dA-tailing was then performed and the TrueSeq DNA LT kits Set A (Illumina, #15041757) was used for adapter ligation and PCR amplification. PCR primers were removed using Ampure XP beads. The Hi-C libraries were sequenced using 50 bp paired end reads on Illumina HiSeq4000.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: The cohesin complex plays an essential role in chromosome maintenance and transcriptional regulation. Recurrent somatic mutations in the cohesin complex are frequent genetic drivers in cancer including myelodysplatic syndromes (MDS) and acute myeloid leukemia (AML). Here, using genetic dependency screens of STAG2-mutant AML, we identified DNA damage repair and replication as genetic dependencies in cohesin-mutant cells. We demonstrated increased levels of DNA damage and sensitivity of cohesin-mutant cells to PARP inhibition. We developed a mouse model of MDS in which Stag2 mutations arise as clonal secondary lesions in the background of clonal hematopoiesis driven by Tet2 mutations, and demonstrated selective depletion of cohesin-mutant cells with PARP inhibition in vivo. Finally, we demonstrated a shift from STAG2- to STAG1-containing cohesin complexes in cohesin-mutant cells, which is associated with longer DNA loop extrusion, more intermixing of chromatin compartments, and increased interaction with PARP and RPA proteins. Our findings inform the biology and therapeutic opportunities for cohesin-mutant malignancies. Overall design: Hi-C was performed in a set of STAG2-wild type (2 clones) and knockout AML cell line (2 clones). We observed a global weakening of compartmentalization (A and B compartments), a decrease in TAD boundary insulation and an increase in the loop size in the STAG2 knockout cells.
SAMPLE_TITLE: ZT-KO2-STAG2-A2-H4-R2-T1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "AML U937 cell line STAG2 KO"}, {"TAG": "enzyme", "VALUE": "DpnII"}, {"TAG": "cell line", "VALUE": "U937"}]
Pool_Member_@attributes_sample_title: ZT-KO2-STAG2-A2-H4-R2-T1
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