EXPERIMENT_TITLE: GSM3384491: Clone 27b - mitotic; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: In-situ Hi-C experiments were performed as previously described (Rao et al., 2014). Cells were fixed with 1% of formaldehyde at room temperature for 10 minutes. After inactivating with glycine, crosslinked cell pellets were washed with cold PBS once before snap freezing with liquid nitrogen. 1-3 million crosslinked cells were used per Hi-C experiment as follows: Briefly, the crosslinked cell pellets were thawed on ice, lysed on ice for 15 minutes (lysis buffer: 10 mM Tris, pH 8.0, 10 mM NaCl, 0.2% IGEPAL CA-630 with proteinase inhibitor) followed by a washing with lysis buffer. The pellets were then resuspended in 50 µl 0.5% of SDS and incubated at 62°C for 10 minutes. Permeabilization was quenched by adding 25 µl 10% Triton X-100 and 145 µl water, and incubation at 37°C for 15 minutes. Digestion was performed overnight at 37°C, shaking at 1,000 rpm with 1xNEBuffer 2 and 100 units of MboI, followed by inactivation of MboI at 62°C for 20 minutes. Biotin fill-in reaction was performed for 1.5 hours at 37°C with 15 nmol of dCTP, dGTP, dTTP, biotin-14-dATP each and 40 unit of Klenow. In-situ ligation was then performed at room temperature with slow rotation in a total volume of 1.2 ml containing 1xT4 ligase buffer, 0.1mg/ml BSA, 1% Triton X-100 and 4000 unit of T4 ligase. After ligation, the DNA was reverse crosslinked at 55°C for 30 minutes followed by 68°C overnight in the presence of SDS, NaCl and proteinase K. Afterwards, DNA was extracted by ethanol precipitation and then sheared to an average of 400bp with Covaris M220. Ampure beads were used to further purify the sheared DNA and select the fragments between 300 to 700bp. The biotin pull-down enrichment and library construction were performed as previously described (Rao et al., 2014).
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Gene amplification leading to increased oncogene expression or anticancer drug resistance is a frequent event in cancer. Cytogenetic, genomic, and transcriptomic analyses were used to determine the initiating events of gene amplification in a tunable cell culture system developing resistance to chemotherapy. Chromosome shattering (chromothripsis) was found to be a major resistance driver through the production of extra-chromosomal double minutes either directly or subsequent to formation of a homogenous staining region (HSR). Formation and maintenance of double minutes were enhanced by PARP and DNA-PK activities. Double minutes were found to be highly dynamic, undergoing structural evolution to produce increased drug tolerance or re-integration into ectopic chromosomes under conditions of DNA damage. Genome rearrangement profiles produced through acquired drug resistance are similar to human cancer examples with oncogene amplification. Thus, chromothripsis is a driver of genome evolution that enables rapid acquisition of tolerance to altered growth conditions. Overall design: Examination of DNA interactions using in situ Hi-C in two different cells (with double minutes or with HSRs) and in control cells
SAMPLE_TITLE: Clone 27b - mitotic
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "HeLa clone"}, {"TAG": "resistance", "VALUE": "Na\u00efve cells"}, {"TAG": "amplification type", "VALUE": "none"}, {"TAG": "cell cycle stage", "VALUE": "mitotic"}]
Pool_Member_@attributes_sample_title: Clone 27b - mitotic
RUN_SET_RUN_Pool_Member_@attributes_sample_title: Clone 27b - mitotic
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