| Construction Protocol | Primary aortic endothelial cells (ECs) isolated from human aorta (HAECs: two 21 year old Caucasian males), mouse aorta (MAECs: two biological replicates of C57BL/6 males from pooled mice), and bovine aorta (BAEC: two biological replicates) were grown in cell culture. Cells 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 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). ~50K cells were harvested from each biological replicate of un-stimulated and TNF-α stimulated (45 min. 10 ng/ml) HAECs (#1487 and #2139), MAECs (#A092913T2MP and #B092913T2MP), and BAECs (#1165 and #1190). Cells were lysed in nonionic detergent lysis buffer (10 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 10 mM NaCl, 0.1% NP-40) for nuclei isolation. The nuclei were pelleted and re-suspended in 50 ul transposition reaction solution of 2.5 ul transposome complex containing Tn5 transposase attached to sequencing adapters (Illumina Nextera Tagment DNA enzyme TDE1, Nextera® DNA Sample Preparation Kit cat# FC-121-1030), 25 ul Illumina Tagment DNA buffer (TD, Nextera® DNA Sample Preparation Kit cat# FC-121-1030), and 22.5 ul of nuclease free water and incubated at 37°C for 30 min under gentle mixing condition (300 rpm). The transposed and adapter-ligated DNA fragments were purified using a Qiagen MinElute PCR Purification Kit The purified DNA was PCR amplified using barcoded primers (Nextera Index Kit, FC-121-1011) for 12 cycles: [72°C for 5 min, 98°C for 30 sec (98°C for 10 sec, 63°C for 30 sec, 72°C for 1 min) x 12]. Agencourt AMPure XP beads (Beckman Coulter) were used for size-selection of >150bp DNA fragments. |
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