Illumina HiSeq 2500 paired end sequencing; ATAC-seq of primary mammalian aortic endothelial cells before and after TNFα stimulation
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
ATAC-Seq
Antigen
ATAC-Seq
Cell type
Cell type Class
Cardiovascular
Cell type
HAEC
Primary Tissue
Aorta
Tissue Diagnosis
Normal
Attributes by original data submitter
Sample
ENA first public
2020-04-14
ENA last update
2019-04-16
ENA-CHECKLIST
ERC000011
External Id
SAMEA5568456
INSDC center alias
The Hospital for Sick Children, Genetics and Genome Biology University of Toronto, Department of Molecular Genetics
INSDC center name
The Hospital for Sick Children, Genetics and Genome Biology University of Toronto, Department of Molecular Genetics
INSDC first public
2020-04-14T16:26:05Z
INSDC last update
2019-04-16T11:56:50Z
INSDC status
public
Submitter Id
E-MTAB-7878:HAEC_ATAC_control_biorep_1
age
21 years old
broker name
ArrayExpress
cell type
primary human aortic endothelial cell
common name
human
individual
1487
organism part
aorta endothelium
replicate
1
sample name
E-MTAB-7878:HAEC_ATAC_control_biorep_1
sex
male
stimulus
control
Sequenced DNA Library
library_name
HAEC_ATAC_control_biorep_1_p
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
PCR
library_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.