EXPERIMENT_TITLE: GSM2486811: HUVEC TCC hypoxia replicate 1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: 90 million human umbilical vein endothelial were fixed with 1 % PFA/PBS in room temperature for 10 minutes and crosslinking was stopped by incubating cells 5 min in presence of 125 mM glycine. Cells were washed two times with ice-cold PBS containing 0.5 % BSA and scraped into lysis buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 5 mM MgCl2, 0.2 % NP-40 and protease inhibitor cocktail (Roche). Samples were vortexed, incubated on ice for 10 min, centrifugated for 5 minutes in 1500 g in +4 °C and washed once again with lysis buffer. Cells were washed twice with ice-cold wash buffer containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl and 0.5M EDTA and nuclei were isolated by incubating samples 10 minutes at +65 °C in wash buffer with 0.55 % SDS and collected by sentrifugation (1000 g for 1 min). Later steps followed the protocol described (Kalhor et al., 2012), except MboI (NEB) was used to digest chromatin, T1 Streptavidin dynabeads (Invitrogen) were used in all the pulldown steps, NucleoSpin Gel & PCR Clean-up kit (Macherey-Nagel) for clean-up steps and Bioruptor NextGen sonicator for DNA shearing (High setting, 30s/30s cycles for total 35 cycles). Libraries were amplified in 15 PCR cycles, size selected for 225 – 550 bp range on 10 % TBE-gel (Invitrogen). Gel was grinded and soaked with 0.1 % Tween-20 and 150 mM NaCl and DNA was eluted through Ultrafree Centrifugal filter column (Millipore), cleaned up with ChIP DNA clean & concentrator kit (Zymo Research) and paired-end sequenced with Illumina HiSeq 2000.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Endothelial cells, which constitute the luminal layer of blood and lymphatic vessels, differentiate from mesodermal progenitors during a process called vasculogenesis. By analyzing changes in chromatin interactions in human umbilical vein endothelial cells (HUVEC) compared to human embryonic stem cells (ESC) and mesendoderm cells (MSC), we identified regions exhibiting endothelial-specific compartmentalization and changes in the degree of connectivity within topologically associated domains (TADs). These EC-specific regions of chromatin interactions were characterized by EC-specific transcription, binding of lineage-determining transcription factors and cohesin. In addition, we developed a method that identified for the first time hundreds of cell-type specific long-range interactions between TADs. Most of these interactions were connections between regions enriched for H3K9me3, suggesting that majority of long-range interactions form between regions of heterochromatin involving pericentromeric regions during cellular differentiation. Secondly, we demonstrate that the hierarchy of chromatin domains is already pre-established in ESCs suggesting that higher level chromatin aggregation is conserved throughout cellular differentiation. Finally, we investigated the effect of hypoxia on transcription and chromatin interactions of ECs. Although hypoxic stress altered the expression of hundreds of genes, it had only a minimal effect on the chromatin compartmentalization, on the amount of local interactions within TADs or long range interactions. Collectively our results show that large structural rearrangements establish chromatin architecture required for functional endothelium and this architecture remains mostly unchanged in response to hypoxic stress. Overall design: Chromatin interactions of HUVECs were analyzed genome-wide using tethered conformation capture (TCC) in normoxia and hypoxia (8h, 1% O2). Transcriptional response was studied using global run-on sequencing (GRO-seq), cohesin binding using ChIP-seq for RAD21 and histone modifications using ChIP-seq for H3K9me3 and H3K27me3.
SAMPLE_TITLE: HUVEC TCC hypoxia replicate 1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "human umbilical vein endothelial cells"}, {"TAG": "cell type", "VALUE": "human umbilical vein endothelial cells (HUVEC)"}]
Pool_Member_@attributes_sample_title: HUVEC TCC hypoxia replicate 1
RUN_SET_RUN_Pool_Member_@attributes_sample_title: HUVEC TCC hypoxia replicate 1
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