EXPERIMENT_TITLE: GSM7697638: MB164, G3b, Hi-C; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Frozen tissues were pulverized using a mortar and pestle kept cold on a bed of dry ice into a fine powder. The tissue powder was then transferred to a 15mL conical tube containing 5mLs of DPBS and fixed with 2% formaldehyde for 10 minutes. The fixation was quenched by addition of 0.2M Glycine. The fixed tissue was pelleted by centrifugation, washed 1x with DPBS, and then flash frozen until ready for further processing. For Hi-C experiments, the fixed frozen tissue pellets were first resuspended in 3mLs of lysis buffer (10mM Tris-HCl pH 8.0, 5mM CaCl2, 3mM MgAc, 2mM EDTA, 0.2mM EGTA, 1mM DTT, 0.1mM PMSF, 1X Complete Protease Inhibitors). The sample was transferred to an M-tube and dissociated using a GentleMACS Tissue dissociator (Miltenyi) using the “Protein M-tube” setting. The sample was removed from the M-tube into a 50mL conical. The M-tube was washed with 3mLs of lysis buffer with 0.4% Triton X-100 added, and this wash was combined with the original 3mLs of sample for a total volume of 6mLs with final concentration of 0.2% Triton X-100. The sample was then passed through a 40µM cell strainer. The strainer was washed with an additional 2mLs of lysis buffer with 0.2% Triton X-100. The sample was then centrifuged and washed with 1mL of lysis buffer with 0.2% Triton X-100. After centrifugation, the sample was resuspended in 0.5% SDS and processed using the MboI enzyme. Libraries were prepared using the Illumina TruSeq LT sequencing adaptors. Initial QC sequencing was first performed on a MiSeq to assess library quality, and if sufficient, was subject to production scale sequencing on the NovaSeq platform, respectively.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Circular extrachromosomal DNA (ecDNA) in patient tumor genomes is an important driver of oncogenic gene expression, evolution of drug resistance, and poor patient outcomes. Applying computational methods for detection and reconstruction of ecDNA across a retrospective cohort of 481 medulloblastoma (MB) tumors from 465 patients, we identify circular ecDNA in 82 patients (18%). Patients with ecDNA+ MB were more than twice as likely to relapse and three times as likely to die within 5 years of diagnosis. Individual tumors harbor multiple ecDNA lineages, each containing distinct amplified oncogenes. Multimodal sequencing, imaging, and CRISPR inhibition experiments in MB models reveal intratumoral heterogeneity of ecDNA copy number per cell and frequent putative "enhancer rewiring" events on ecDNA. This study reveals the frequency and diversity of ecDNA in a subset of highly aggressive MB tumors, and suggests copy number heterogeneity and enhancer rewiring as clinically relevant features of ecDNA in MB. Overall design: Bulk Hi-C of frozen primary medulloblastomas (MB***). Hi-C of fresh patient-derived xenograft (PDX) mouse model (RCMB56). Hi-C of cell line D458.
SAMPLE_TITLE: MB164, G3b, Hi-C
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "medulloblastoma"}, {"TAG": "tissue", "VALUE": "medulloblastoma"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: MB164, G3b, Hi-C
RUN_SET_RUN_Pool_Member_@attributes_sample_title: MB164, G3b, Hi-C
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