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: SAMEA111406957
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: ERS13500824
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12212:MAEC_H3K4me2_control
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: aortic endothelial cell
characteristics.characteristic: cell_type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Primary aortic endothelial cells (ECs) isolated from mouse aorta from mixed male individuals were grown in cell culture. Cells were cross-linked in 1% FA. Cells were then lysed for nuclei isolation. The chromatin was sheared into 100-500 bp DNA fragments by sonication (Misonix Sonicator) at 27-28 Watt for 26 cycles (10 seconds ON, 1 minute OFF, 28 W). We used ~1.7 % of chromatin as input. ChIPs were performed at 4°C overnight with antibodies designed against H3K4me2 and H3K27ac. The cross-links were reversed at 65°C for 16 h. Proteins and RNA were enzymatically digested and DNA was purified using phenol-chloroform extraction and ethanol precipitation. ChIP DNA and 220 ng of input DNA were mixed with 3 units of T4 DNA polymerase (NEBNext® DNA Library Prep Master Mix Set for Illumina, #E6040L) to create blunt ends. To generate 5´-phosphates, 10 units of T4 PNK (NEBNext® DNA Library Prep Master Mix Set for Illumina, #E6040L) were added to the blunt ended DNA in NEBNext End Repair Reaction Buffer (NEB, #E6042A) and incubated for 30 min at room temperature. The DNA was purified using DNA Clean and Concentrator (Zymogen, cat# D4014). To add dAMP to the 3’ ends, the end-repaired DNA was mixed with Klenow Fragment (3’→5’ exo-) (NEB, #E6044A) in NEBNext dA-Tailing Reaction Buffer (NEB, #E6045A) and incubated for 40 min at 37°C. The dA-tailed DNA was then purified. Illumina sequencing adapters containing uracil hairpin loop structure and 3’ T overhangs (NEB, #E7337A) were ligated to the DNA by adding 150 units of Quick T4 DNA Ligase (NEB, #E6047A) in Quick Ligation Reaction Buffer (NEB, #E6048A) and incubation for 15 minutes at room temperature. Looped adapter sequences were opened by removal of uracil from hairpin structures by adding 3 units of USER enzyme (Uracil-Specific Excision Reagent) (NEB, M5505S) and incubation at 37°C for 15 min. This made DNA accessible for PCR amplification with barcoded primers for Illumina sequencing (NEB, cat# E7335L). PCR amplifications were carried out for 16 cycles [98°C 30 sec, (98°C 10 s, 65°C 30 s, 72°C 30 sec) x 17 cycles, 72°C 5 min, 4°C hold]. The amplified and barcoded library was then selected for 200-350 bp fragments using Pippin Prep (Sage Science)
characteristics.characteristic: description
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: aorta endothelium
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.characteristic: sex
characteristics.text: C57BL/6
characteristics.characteristic: strain
characteristics.text: MAEC_H3K4me2_control
characteristics.characteristic: title