EXPERIMENT_TITLE: GSM4663834: HiC_MRC5; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: The crosslinked cell suspension was split and centrifuged at 800xg for 10 min. The supernatant was discarded by aspiration. The fixed cells were resuspended in 1 ml of lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630, 1/10 vol. of proteinase inhibitor cocktail (Sigma)) and then incubated on ice for 20 min. Nuclei were pelleted by centrifugation at 4°C and 600 g for 5 min and then washed with 1 ml of lysis buffer, followed by another centrifugation under similar conditions. After washing twice with restriction enzyme buffer, the nuclei were resuspended in 400 μl of restriction enzyme buffer and transferred to a safe-lock tube. Next, the chromatin was solubilized with dilute SDS and incubated at 65°C for 10 min. After quenching the SDS by Triton X-100 overnight, digestion was performed with 4 cutter restriction enzymes (400 units MboI) at 37°C on a rocking platform. The proximal chromatin DNA was religated by the ligation enzyme. The nuclear complexes were reversed crosslinked by incubating with proteinase K at 65°C. DNA was purified by phenol-chloroform extraction. Biotin-C was removed from nonligated fragment ends using T4 DNA polymerase. Fragments were sheared to a size of 200-600 base pairs by sonication. The fragment ends were repaired by a mixture of T4 DNA polymerase, T4 polynucleotide kinase and Klenow DNA polymerase. Biotin-labelled HiC samples were specifically enriched using streptavidin C1 magnetic beads. The fragment ends were adding by A-tailing by Klenow(exo-) and then adding Illumina paired-end sequencing adaptor by ligation mix. Finally, the Hi-C libraries were amplified using 12-14 cycles of PCR and sequenced on the Illumina HiSeq platform. Sequencing interacting partners were obtained by an Illumina HiSeq instrument with 2×150-bp reads.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Small cell lung cancer (SCLC) is an aggressive form of lung cancer withunique change in chromosomal structure while the basis of the aberrant gene expression in SCLC remains largely unclear. Topologically associated domains (TADs) are structural and functional units of the human genome. Genetic and epigenetic alterations in the cancer genome can lead to disruption of TAD boundaries and may cause gene dysregulation. To understand thepotentialregulatoryroleofthisprocess in SCLC, we developed TAD boundary alteration-related gene identification in tumour (TARGET), a computational framework that,forthefirsttime, enables a systematic identificationof candidate dysregulated genes associated with altered TAD boundaries. Using TARGET to compare gene expression profiles between SCLC and normal human lung fibroblast celllines, we have identified over one hundredgenes in this catagory, amongwhich 24 were further verified in SCLC patient samples using NanoString. Further, analysis revealed a synergistic chromatin structure alteration at both the AB compartment level and TAD boundary level that underlie aberrant gene expression in SCLC. Therefore, TARGET serves as a novel and powerful method could be used to explore the relationship of chromatin structure alteration and gene dysregulation relatedto tumorigenesis, progression, and prognosis of SCLC. Overall design: Examination chromatin structure and gene expression in  two human lung carcinoma cell lines and fibroblast cell
SAMPLE_TITLE: HiC_MRC5
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "HiC_MRC5"}, {"TAG": "cell line", "VALUE": "MRC5"}, {"TAG": "cell type", "VALUE": "Normal lung fibroblast cell"}]
Pool_Member_@attributes_sample_title: HiC_MRC5
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