EXPERIMENT_TITLE: GSM3593257: HiC_teloHAEC_0h_r2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: In situ Hi-C libraries were prepared as previously described (Belaghzal, Decker and Gibcus, 2017) with the following specifications and modifications:  Approximately 8x10^6 cells per sample were crosslinked (PFA 1%), pelleted and washed in ice-cold PBS prior to lysis and chromatin digestion with DpnII. Biotin-14-dATP fill-in and blunt end ligation was performed as described. Reverse crosslinking was performed in two subsequent 16 and 2 hours incubations with 500 μg of proteinase K prepared at 10 mg/mL in 5mM Tris-HCl pH 7.5, 50% glycerol, 1mM CaCl2 for each step. DNA purification was performed using 15mL MaXtract High Density tubes (Qiagen) with subsequent biotin removal from un-ligated ends using T4 DNA polymerase. For NGS preparation, between 20 and 40 μg of purified Hi-C DNA was used as starting material for all subsequent steps. Sonication to 200-300 bp fragments was carried in an S2 Focused-ultrasonicator with no alterations to the suggested parameters. 300-500 bp fragments were purified with AMPure XP beads and end-repaired as described. Biotin pulldown was performed with 200 μg of Dynabeads MyOne Streptavidin C1 (Invitrogen) per sample. A-tailing was performed as described. Illumina adapters were ligated to 3' d-ATP overhangs using T4 DNA ligase. Production PCR was carried out with 9 cycles of PCR to obtain sufficient quantity for NGS while limiting PCR duplicates. Primers and primer dimers were removed by purification AMPure XP beads.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Genome-wide association studies (GWAS) have identified 100s of loci associated with coronary artery disease (CAD) and blood pressure (BP)/hypertension. Many of these loci are not associated with traditional risk factors, nor include obvious candidate genes, complicating their functional characterization. We hypothesized that many GWAS loci associated with vascular diseases modulate endothelial functions. Endothelial cells play critical roles in regulating vascular homeostasis (e.g. selective barrier, inflammation, hemostasis, vascular tone) and endothelial dysfunction is a hallmark of atherosclerosis and hypertension. We generated an integrated map of gene expression (RNA-sequencing), open chromatin regions (ATAC-sequencing), and 3D interactions (Hi-C) in resting and TNFa-treated human endothelial cells. We showed that genetic variants associated with CAD and BP are enriched in open chromatin regions identified in endothelial cells. We used physical loops identified by Hi-C to link open chromatin peaks that include CAD or BP SNPs with the promoter of genes expressed in endothelial cells. This analysis highlighted 4,548 combinations of regulatory elements-promoters, including 108 pairs that involve a differentially open chromatin site and a differentially expressed gene following TNFa treatment. At a CAD locus, we validated one of these pairs by engineering a deletion of the TNFa-sensitive regulatory element using CRISPR/Cas9 and measuring an effect on the expression of the novel CAD candidate gene AIDA. Our data support an important role played by genetic variants acting in the vascular endothelium to modulate inter-individual risk in CAD or hypertension Overall design: Hi-C of TNF-alpha treated endothelial cells (teloHAEC) for 0h or 4h
SAMPLE_TITLE: HiC_teloHAEC_0h_r2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Endothelial Cells"}, {"TAG": "cell type", "VALUE": "immortalized Human Aortic Endothelial Cells"}, {"TAG": "passages", "VALUE": "passages 23 to 25"}, {"TAG": "tnf-alpha treatment", "VALUE": "0h"}]
Pool_Member_@attributes_sample_title: HiC_teloHAEC_0h_r2
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