Histone ChIP-seq of primary mouse aortic endothelial cells treated with TNFα against untreated controls
Histone ChIP-seq of primary mouse aortic endothelial cells treated with TNFα against untreated controls
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)