EXPERIMENT_TITLE: H3K4me3 HiChIP of immortalized human aortic endothelial cells treated with TNFα against untreated controls
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: TeloHAECs were grown in cell culture. 5 million TeloHAECs per replicate were crosslinked in 37% formaldehyde as per instructions of the Arima Hi-C kit (cat# A510008) and frozen down as 106 cells per aliquot. After DNA estimation, cells corresponding to 3 ug of total DNA were thawed on ice and lysed in 20 ul of cold lysis buffer for 15 min, followed by permeabilization, digestion, and proximity ligation as per the instructions of the Arima Hi-C kit (cat# A510008).We skipped step 10 of the Enzyme D step in the Arima kit and proceeded to ChIP. For ChIP, we  adapted the protocol from Fang et al. 2016 with slight modifications. After proximity ligation, the nuclei were spun down at 2,500 g for 5 min at 4°C and the supernatant was discarded. The nuclei were then resuspended in 130 μl RIPA buffer (10 mM Tris, pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate) with proteinase inhibitors. The nuclei were lysed on ice for 10 min and then sonicated using Diagenode Bioruptor® Pico with the following setting: 10 Cycles of 30 Secs On/Off. After sonication, 370 ul of RIPA buffer was added to the samples before they were cleared by centrifugation at 20,000 G for 20 min and the supernatant was collected. The clear cell lysate was mixed with 500ul of antibody (2.5 μg of H3K4me3, Millipore cat #04-745) coated Dynabeads M-280 (Sheep anti-Rabbit Thermo Scientific cat #11201D) and rotated at 4°C overnight. Next day, the beads were collected by centrifugation at 2,000 rpm for 1 min and then resuspended in 1 ml of RIPA buffer and washed for three times in 1x RIPA buffer, twice in high-salt RIPA buffer (10 mM Tris, pH 8.0, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate), once with LiCl buffer (10 mM Tris, pH 8.0, 150 mM LiCl, 1 mM EDTA, 0.5% IGEPAL CA-630, 0.1% sodium deoxycholate), and twice with TE buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA). Washed beads were first treated with 10 μg Rnase A in extraction buffer (10 mM Tris, pH 8.0, 350 mM NaCl, 0.1 mM EDTA, 1% SDS) by shaking at 1000 rpm for 1 h at 37°C. Then 20 μg proteinase K was added and reverse crosslinking was performed by shaking at 1000 rpm overnight at 65°C or at least 2 h. The fragmented DNA was purified with DNA Concentrator (Zymo Research, cat# D4014). Lastly, the biotin pull-down procedure was performed according to Swift Biosciences Accel-NGS 2S Plus DNA Library Kit (cat# 21024) An optimal number of PCR cycles were determined by qPCR using the KAPA library quantification kit (cat# KK4824). Libraries were checked on a bioanalyzer for quality and then sequenced
STUDY_DESCRIPTOR_STUDY_ABSTRACT: TNFa is a potent cytokine to mediate inflammatory response by activation of the master transcription factor NF-kB. Endothelial cells are important participants in inflammatory responses in animals. NF-kB is a major mediator to activate endothelial cells by inducing multiple pro-inflammatory genes in response to TNFa. The process of transcriptional activation is dependent on the context of chromatin interactions/loops. However, the chromatin conformation in response to the cytokine challenge TNFa in human aortic endothelial cells has not been described. Our approach characterized chromatin interactions from active enhancers to promoters as well as active promoters to promoters on a genome-wide scale and highlighted important enhancer candidates for target genes.
SAMPLE_TITLE: TeloHAEC_H3K4me3_TNFa_rep2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "ENA-FIRST-PUBLIC", "VALUE": "2023-03-01"}, {"TAG": "ENA-LAST-UPDATE", "VALUE": "2023-03-01"}, {"TAG": "External Id", "VALUE": "SAMEA111407328"}, {"TAG": "INSDC center alias", "VALUE": "1 Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada 2 Genetics and Genome Biology, Sickkids Research Institute, Toronto, ON, Canada"}, {"TAG": "INSDC center name", "VALUE": "1 Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada 2 Genetics and Genome Biology, Sickkids Research Institute, Toronto, ON, Canada"}, {"TAG": "INSDC first public", "VALUE": "2023-03-01T00:16:59Z"}, {"TAG": "INSDC last update", "VALUE": "2023-03-01T00:16:59Z"}, {"TAG": "INSDC status", "VALUE": "public"}, {"TAG": "Submitter Id", "VALUE": "E-MTAB-12213:TeloHAEC_H3K4me3_TNFa_rep2"}, {"TAG": "broker name", "VALUE": "ArrayExpress"}, {"TAG": "cell_line", "VALUE": "TeloHAEC"}, {"TAG": "cell_type", "VALUE": "aortic endothelial cell"}, {"TAG": "common name", "VALUE": "human"}, {"TAG": "disease", "VALUE": "normal"}, {"TAG": "organism part", "VALUE": "aorta endothelium"}, {"TAG": "sample name", "VALUE": "E-MTAB-12213:TeloHAEC_H3K4me3_TNFa_rep2"}, {"TAG": "scientific_name", "VALUE": "Homo sapiens"}, {"TAG": "sex", "VALUE": "female"}, {"TAG": "stimulus", "VALUE": "tumor necrosis factor alpha; 10 nanogram per milliliter"}]
Pool_Member_@attributes_sample_title: TeloHAEC_H3K4me3_TNFa_rep2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: TeloHAEC_H3K4me3_TNFa_rep2
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