EXPERIMENT_TITLE: GSM5725808: HEK293T, Hi-C, overexpressing NANOG WT, biological replicate 1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Hi-C 3.0 was performed based on a recent protocol, which is largely modified based on in situ Hi-C. Briefly, ~5 million cells were washed once with PBS to remove debris and dead cells, trypsinized off the culture plates, and were cross-linked using 1% formaldehyde for 10mins at room temperature, quenched with 0.75M Tris-HCl pH 7.5 for 5mins. These cells were further cross-linked with 3mM disuccinimidyl glutarate (DSG) for 50mins, and again quenched with 0.75M Tris-HCl pH 7.5 for 5mins. Cross-linked cell pellets were washed with cold PBS, and then resuspended in 0.5 ml ice-cold Hi-C lysis buffer (10 mM Tris-HCl, pH 8.0; 10mM NaCl, 0.2% NP-40 and protease inhibitor cocktail), and rotated at 4°C for 30 mins. Nuclei were washed once with 0.5 ml ice-cold Hi-C lysis buffer. After pelleting down the nuclei, 100 μl of 0.5% SDS was used to resuspend and permeabilize the nuclei at 62 °C for 10 mins. Then 260 μl H20 and 50 μl 10% Triton-X100 were added to quench the SDS at 37 °C for 15 mins. Subsequently, enzyme digestion of chromatin was performed at 37 °C overnight by adding an additional 50 μl of 10X NEB buffer 2, 100U MboI (NEB, R0147M) and 100U DdeI (NEB, R0175L). After overnight incubation, the restriction enzyme was inactivated at 62 °C for 20 mins. To fill-in the DNA overhangs and add biotin, 35U DNA polymerase I (Klenow, NEB, M0210) together with 10 μl of 1mM biotin-dATP (Jeana Bioscience), 1μl 10mM dCTP/dGTP/dTTP were added and incubated at 37°C for 1 hour with rotation. Blunt end Hi-C DNA ligation was performed using 5000 U NEB T4 DNA ligase with 10X NEB T4 Ligase buffer with 10mM ATP, 90 μl 10% Triton X-100, 2.2 μl 50mg/ml BSA at room temperature for four hours with rotation. After ligation, nuclei were pelleted down and resuspended with 440 μl Hi-C nuclear lysis buffer (50mM Tris-HCl pH7.5, 10mM EDTA, 1% SDS and protease inhibitor cocktail), and further sheared using the parameter of 10/20 secs ON/OFF cycle, 25% Amp, 4 mins by a QSonica 800R sonicator. Around 10% of the sonicated chromatin was subjected to overnight decrosslinking at 65°C, protein K treatment, and DNA extraction. After DNA extraction, biotin labelled Hi-C 3.0 DNAs were purified by 20 μl Dynabeads® MyOne™ Streptavidin C1 beads (Thermo Fisher 65002). The biotinylated-DNA on C1 beads was used to perform on-beads library making with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (NEB, E7645L) following manufacturer's instructions. The sequencing was done on a NextSeq 550 platform with PE40 mode.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: NANOG is a key stem cell pluripotency factor. NANOG's unique ability to form prion-like assemblies provides a cooperative and concerted DNA bridging mechanism essential for chromatin reorganization and dose-sensitive activation of ground state pluripotency. We take advantage of Hi-C and ChIP-seq experiments to directly address whether NANOG can bridge DNA elements together in human cells, and our results support this. Overall design: NANOG function in DNA bridging in human cells.
SAMPLE_TITLE: HEK293T, Hi-C, overexpressing NANOG WT, biological replicate 1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Kidney"}, {"TAG": "cell line", "VALUE": "HEK293T"}, {"TAG": "overexpression", "VALUE": "NANOG WT"}, {"TAG": "treatment", "VALUE": "untreated"}]
Pool_Member_@attributes_sample_title: HEK293T, Hi-C, overexpressing NANOG WT, biological replicate 1
RUN_SET_RUN_Pool_Member_@attributes_sample_title: HEK293T, Hi-C, overexpressing NANOG WT, biological replicate 1
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