EXPERIMENT_TITLE: GSM6725234: sTL-CMs_Rep2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Cultured nTL-CMs and sTL-CMs at Day30 were collected and processed for Hi-C. Hi-C libraries for normal and short telomere hiPSC-CM were constructed with previously described in situ Hi-C protocols with minor modifications4. Cells were fixed with 0.92 mL of 1% v/v formaldehyde at RT for 10 min. Next, 0.08mL of 2.5 M glycine was added. The samples were incubated at RT for 5 min and on ice for at least 15 min to quench crosslinking completely. The fixed samples were collected by centrifuging and then lysed with 1 mL of ice-cold lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA630) with protease inhibitors subsequently. The samples were incubated on ice for 30 min and centrifuged for 5 min at 3,000 ×g at 4°C to collect the nuclei. The nuclear pellets were washed once with NEBuffer 3.1, then resuspended in 20 μL of 10× NEBuffer 3.1, 140 μL of ddH2O, and 40 μL of 0.5% SDS; and then incubated at 65°C for 5 min to open up chromatin. Next, 22 μL of 10% Triton X-100 (Sigma) was added before incubation at 37°C for 15 min to quench SDS. 5 μL of 10× NEBuffer 3.1, 15 μL of ddH2O, and 8 μL (400 U) of DpnII (NEB) were added to the samples to digest chromatin. Digestion was performed overnight at 37°C on a thermomixer with interval shaking. The samples were incubated at 65°C for 20 min to inactivate the restriction enzyme. To fill the restriction overhangs, 60 μL of fill-in mix (37.5 μL of 0.4 mM biotin-14-dATP (Life Technologies), 1.5 μL of 10 mM dCTP/dGTP/dTTP, 10 μL of 5 U/μL Klenow, 6 μL of 10× NEBuffer 3.1, and 2 μL of Milli-Q water) was added to the reaction, and the samples were incubated at 25°C for 4 h. Ligation mix (498 μL of water, 240 μL of 5× T4 DNA ligase buffer (Invitrogen), 100 μL of 10% Triton X-100, 12 μL of 10 mg/mL BSA., and 50 μL of 1 U/μL T4 DNA ligase (Invitrogen) were then added into the samples. The samples were incubated at 16°C for exactly 4 h. After ligation, the samples were centrifuged and then resuspended in 500 μL of 1× NEBuffer. To reverse crosslinking, 30 μL of 20 mg/mL proteinase K (NEB) and 50 μL of 10% SDS (Sigma) were added to the samples, and the samples were then incubated at 65°C overnight. Phenol-chloroform extraction and ethanol precipitation were performed. The purified DNA was sheared with a Covaris M220. End repair of DNA fragments and ligation with adapters were performed using the End Repair/dA-Tailing Module (NEB) and the Ligation Module (NEB) according to the product manuals after DNA shearing. To purify biotinylated DNA fragments, Dynabeads MyOne Streptavidin T1 beads (Life Technologies) were used to bind the biotinylated DNA. The beads were washed twice with 500 μL of 1× TWB on a Thermomixer at 55°C for 2 min with mixing and resuspended in 50 μL of Milli-Q water. The samples were amplified with Phusion High-Fidelity DNA Polymerase (Thermo Fisher) for 6-8 cycles. AMPure XP beads (Beckman Coulter) were agopted for size selection.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Telomeres, TTAGGGn DNA repeat sequences located at the ends of eukaryotic chromosomes, play a pivotal role in ageing and are targets of DNA damage and DNA damage response. Although short telomeres have been implicated in heart failure, little is known about the role of telomeres in cardiomyocytes.To fill this gap in knowledge, we studied myocardial ageing in the form of telomere lengths under isogenic background. Overall design: In this study, the dynamics of chromatin structure of nTL-CMs and sTL-CMs were depicted.
SAMPLE_TITLE: sTL-CMs_Rep2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "cardiomyocytes"}, {"TAG": "cell_type", "VALUE": "hiPSCs-derived cardiomyocytes"}, {"TAG": "treatment", "VALUE": "BIBR"}]
Pool_Member_@attributes_sample_title: sTL-CMs_Rep2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: sTL-CMs_Rep2
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