title: Illumina HiSeq 2500 sequencing; Multi-species RELA ChIP-seq of mammalian aortic endothelial cells before and after TNFα stimulation
design: Multi-species RELA ChIP-seq of mammalian aortic endothelial cells before and after TNFα stimulation
library_construction_protocol: Primary aortic endothelial cells (ECs) isolated from human aorta (HAECs: two 21 year old Caucasian males; a 15 year old male; and a 60 year old male), mouse aorta (MAECs: two biological replicates of C57BL/6 males pooled from multiple mice), and bovine aorta (BAEC: two biological replicates of unknown sex) were grown in cell culture.  HAECs, MAECs, BAECs, and TeloHAECs (ATCC CRL-4052) were cultured at 37°C and 5% CO2 supplied with Endothelial Cell Growth Media MV2 (PromoCell) supplemented with 5% fetal calf serum (PromoCell), 5 ng/ml recombinant human epidermal growth factor (PromoCell), 0.5 ng/ml recombinant human vascular endothelial growth factor 165 (PromoCell), 10 ng/ml recombinant human basic fibroblast growth factor (PromoCell), 20 ng/ml long R3 insulin-like growth factor-1 (PromoCell), 1 μg/ml ascorbic acid (PromoCell) and 0.2 μg/ml hydrocortisone (PromoCell). All experiments were carried out before passage 9. HAECs and TeloHAECs were treated with 10 ng/mL recombinant human TNFα (Cell Applications, cat# RP1111-50), MAECs were treated with 10 ng/mL recombinant mouse TNFα (Cell Applications, cat# RP2031-20), and BAECs were treated with 10 ng/mL recombinant bovine TNFα (R&D Systems, cat# 2279-BT-025) for 45 mins in basal Endothelial Cell Growth Media MV2 (PromoCell) without supplements. The un-stimulated control samples were treated with vehicle (water). For RELA ChIP, cells were initially cross-linked in 2mM disuccinimidyl glutarate (DSG) in PBS under shaking conditions for 30 min followed with the 1% FA (formaldehyde) cross-linking for 10 min at room temperature. For JUN and histone modification ChIPs, 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 RELA (Santa Cruz Biotechnology cat# sc-372, Abcam cat# ab7970), JUN (Santa Cruz Biotechnology, H-79 cat# sc-1694), H3K27ac (EMD Millipore cat# 05-1334), H3K4me2 (EMD Millipore cat# mp07-030), H3K4me3 (EMD Millipore cat# 17-678), H3K4me1 (Abcam cat# ab8895), and H3K27me3 (EMD Millipore cat# 07-449). 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)