EXPERIMENT_TITLE: GSM3438652: TNF_rep1 [Hi-C]; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: In-situ Hi-C was performed in two replicates for HUVECs treated with or without TNF-α for 60 min. Cells were cross-linked for 10 min at room temperature with 1% formaldehyde and quenched for 5 min at room temperature with 0.2M glycine. The cross-linked cells were centrifuged at 2,500xg for 5 min at 4°C. To isolate nuclei, cross-linked cells were resuspended in 200 μl of lysis buffer (10 mM Tris-HCl (pH 8.0), 10mM NaCl, 0.2% NP40, Protease Inhibitor Cocktail (Sigma-Aldrich)) and incubated on ice for 15 min. The suspension was then centrifuged at 2,500xg for 5 min and the pellet washed by resuspending in 300 μl of lysis buffer and centrifuging at 2,500xg for 5 min at 4°C. The pellet was resuspended in 50 μl of 0.5% SDS and incubated for 10 min at 62°C. After heating, 170 μl of 1.47% TritonX-100 was added to the suspension and incubated for 15 min at 37°C. To digest chromatin, 25 μl of 10X NEBuffer2 and 100U MboI (New England Biolabs, Beverly, MA) was added to the suspension and incubated at 37°C overnight with rotation. Enzymes were inactivated by heating for 20 min at 62°C. Fragmented ends were biotin-labeled by adding 50 μl of mixture containing 0.3 mM biotin-14-dATP (Thermo Fisher Scientific), 0.3 mM dATP (Thermo Fisher Scientific), 0.3 mM dTTP (Thermo Fisher), 0.3 mM dGTP (Thermo Fisher Scientific), and 0.8 U/ul Klenow (Enzymatics, Beverly, MA) and incubated for 1 hr at room temperature with rotation. The biotin-labeled fragmented ends were subsequently ligated by adding 900 μl of mixture containing 120 μl of 10X T4 DNA ligase buffer (New England Biolabs), 100 μl of 10% TritionX-100, 12 μl of 10 mg/ml BSA (New England Biolabs), 5 μl of 400 U/μl of T4 DNA Ligase (New England Biolabs) and 663 μl H2O and incubated for 4 hrs at room temperature with rotation. The nuclei were pelleted for 5 min at room temperature at 2,500xg. After removing supernatant, the nuclei were resuspended in 550 μl of 10 mM Tris-HCl (pH 8.0). The nuclei were reverse-crosslinked by adding 50 μl of Proteinase K (New England Biolabs) and 57 μl of 10% SDS, and incubated for 30 min at 55°C. Subsequently, the nuclei mixed with 67 μl of 5 M NaCl were incubated at 68°C overnight. After cooling samples for 10 min at room temperature, DNA was purified by 0.8X AMPureXP beads (Beckman Coulter Inc., Brea, CA) and eluted in 100 μl of 10 mM Tris-HCl (pH 8.0). DNA shearing was performed using focused-ultrasonicators (Covaris, E220; Power 140 W, Duty factor 10%, Cycle per bust 200, Time 67 sec, temperature 7°C) and the final sample volume after shearing was brought to 200 μl using 10 mM Tris-HCl (pH 8.0). To design DNA selection for 200-600 bp size, 120 μl of AMPureXP beads was added into 200 μl of sample (beads ration: 0.6) and incubated for 5 min at room temperature with inversion. To remove DNA fragments >600 bp, the clear supernatant was collected on magnet and beads were discarded. As a second round of size selection, 80 μl of AMPureXP beads was added into the supernatant and incubated for 5 min at room temperature with inversion. The supernatant was removed on magnet and beads were washed twice with 700 μl of 70% ethanol, dried up for 5 min. DNA fragments (200-600 bp) were eluted from beads by 300 μl of 10 mM Tris-HCl (pH 8.0). For biotin pull down, 100 μl of 10mg/ml Dynabeads My One T1 Streptavidin beads (Thermo Fisher Scientific) was washed with 1X Tween Wash Buffer (5m M Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl, 0.05% Tween). The beads resuspended in 300 μl of 2X Binding Buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2M NaCl) were transferred to sample tubes and incubated for 15 min at room temperature. The beads were subsequently washed twice with 1X Tween Wash Buffer for 2 min at 55°C with mixing and washed once with 1X NEB T4 DNA ligase buffer (Enzymatics). To repair fragmented ends and remove biotin from unligated ends, the beads were resuspended in 85 μl of 1X NEB T4 DNA ligase buffer (Enzymatics), 5 μl of 10 mM dNTP Mix (Thermo Fisher Scientific), 5 μl of 10 U/μl of NEB T4 PNK (New England Biolabs), 4 μl of 3 U/μl NEB T4 DNA Polymerase (New England Biolabs) and 1 μl of 5 U/μl Klenow (Enzymatics), and incubated for 30 min at room temperate. After supernatant was discarded on magnet, the beads were washed twice with 1X Tween Wash Buffer for 2 min at 55°C with mixing and resuspended in 100 μl of 1X NEB Buffer2. For dA-tailing, the beads were resuspended in 90 μl of 1X NEB Buffer2, 5 μl of 10 mM dATP (Thermo Fisher Scientific) and 5 μl of 5 U/μl of Klenow (3'→5' exo-) (Enzymatics), and incubated for 30 min at 37°C. The beads were subsequently washed twice with 1X Tween Wash Buffer as before and once with 1X NEB Quick Ligation Reaction Buffer (New England Biolabs). To ligate adapters, the beads were suspended in 50 μl of 1X NEB DNA Quick Ligation Reaction Buffer, 1 μl of NEXTflex® DNA Barcodes (Bioo Scientific, Austion, TX) and 2 μl of NEB DNA Quick Ligase (New England Biolabs), and incubated for 15 min at room temperature. The beads were washed twice with 1X Tween Wash Buffer as before and once with 10 mM Tris-HCl (pH 8.0), and resuspended in 20 μl of 10 mM Tris-HCl (pH 8.0). After estimating concentration and cycle number for final PCR by qPCR assay, final PCR was directly amplified off the T1 beads using KAPA Hyper Prep Kit (KAPA Biosystems Inc) and DNA was cleaned with 1X AMPure beads, and eluted in 30 μl of 10 mM Tris-HCl (pH 8.0) and sequenced paired-end.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Lysine 9 di-methylation and lysine 27 tri-methylation of histone H3 (H3K9me2 and H3K27me3) are mostly linked to gene repression. However, functions of repressive histone methylation dynamics during inflammatory responses remain poorly understood. Here, we show that lysine demethylase 7A (KDM7A) and 6A (UTX) are rapidly transported to nuclear factor kappa-B (NF-?B) related elements in human endothelial cells in response to tumor necrosis factor (TNF)-a. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and cooperatively activate NF-?B dependent inflammatory genes. Furthermore, using both in situ Hi-C and other 3C based technology, loops between super enhancers (SEs) are newly formed following TNF-a-stimuli at NF-?B-dependent inflammatory loci where KDM7A- and UTX-recruitment coincide. Collectively, these findings suggest that erasing of repressive histone marks by KDM7A and UTX within NF-?B-related elements might functionally associate with formation of SE-SE three-dimensional interactions and could be a cue signal during inflammatory responses in human endothelial cells. Overall design: Total 4 Hi-C samples include [1] no treatment (replicate 1 and 2) [2] TNF-alpha 1 hr (replicate 1 and 2).
SAMPLE_TITLE: TNF_rep1 [Hi-C]
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "human umblical vein endothelial cells"}, {"TAG": "conditions", "VALUE": "TNF 1h"}, {"TAG": "donor", "VALUE": "pooled from multiple donors"}, {"TAG": "passages", "VALUE": "4-7"}, {"TAG": "cell type", "VALUE": "HUVECs"}]
Pool_Member_@attributes_sample_title: TNF_rep1 [Hi-C]
RUN_SET_RUN_Pool_Member_@attributes_sample_title: TNF_rep1 [Hi-C]
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