characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-03-01T00:16:59Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-03-01T00:16:59Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA111407327
characteristics.characteristic: External Id
characteristics.text: 1 Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada 2 Genetics and Genome Biology, Sickkids Research Institute, Toronto, ON, Canada
characteristics.characteristic: INSDC center name
characteristics.text: 2023-03-01T00:16:59Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-03-01T00:16:59Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS13501194
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12213:TeloHAEC_H3K4me3_TNFa_rep1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: TeloHAEC
characteristics.characteristic: cell_line
characteristics.text: aortic endothelial cell
characteristics.characteristic: cell_type
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: 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
characteristics.characteristic: description
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: aorta endothelium
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.characteristic: sex
characteristics.text: tumor necrosis factor alpha; 10 nanogram per milliliter
characteristics.characteristic: stimulus
characteristics.text: TeloHAEC_H3K4me3_TNFa_rep1
characteristics.characteristic: title