|
| Status |
Public on Jun 25, 2019 |
| Title |
ATACseq_HCAEC_24h_r2 |
| Sample type |
SRA |
| |
|
| Source name |
Endothelial Cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human Coronary Artery Endothelial Cells
passages: passages 7 to 9
tnf-alpha treatment: 24h
|
| Treatment protocol |
Endothelial cells were treated with TNF-alpha prepared in culture media at 10 ng/mL for 4h and 24h periods
|
| Growth protocol |
Immortalized human aortic endothelial cells (teloHAEC) were grown in vascular cell basal media supplemented with endothelial cell growth kit-VEGF, 200 U/mL penicillin and 200 μg/mL of streptomycin. Primary human coronary artery endothelial cells (HCAEC) from a single male donor were grown in EGM-2MV supplemented with 200 U/mL penicillin and 200 μg/mL of streptomycin. TeloHAEC and HCAEC (below 3 passages) were maintained under a 5% CO2 atmosphere at 37°C. Endothelial cells were seeded at 2x10^5 cells per well in 6-well plates, grown for 3 days (refreshed media at day 2) until reaching 95-100% confluency and subjected to TNF-alpha treatment.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
5x10^4 cells were spun down at 500g for 5 minutes at 4°C. Whole cell pellets were subjected to a first round of cell membrane lysis using 50 μL of ice-cold hypotonic buffer (0.1% Sodium citrate tribasic dehydrate; 0.1% Triton X-100) and incubating on ice for 30 minutes. The hypotonic buffer was removed by centrifugation at 500g for 5 minutes at 4°C and we subsequently discarded the supernatant. Crude nuclei lysates were prepared by resuspending cells in lysis buffer (10mM Tris-HCl pH 7.4; 10mM NaCl; 3mM MgCl2; 0.1% Igepal CA-630 and incubating for 30 minutes on ice. Following the removal of lysis buffer by centrifugation at 500g for 5 minutes at °4C, transposition reaction of open chromatin was achieved by resuspending free nuclei in tagmentation mix (22.5 μL Tagment DNA Buffer; 2.5 μL Tagment DNA enzyme; 25 μL H2O) (Illumina) and incubating at 37°C for 30 minutes. Purification of DNA was performed with MinElute (Qiagen) according to the manufacturer’s protocol.
Barcoding and amplification was prepared using Nextera Index Kit (Illumina, FC-121-1011) with the following thermal profile: 30 seconds at 98°C and a three-step cycle of 10 seconds at 98°C, 30 seconds at 63°C and 1 minute at 72°C repeated 12 times followed by 5 minutes at 72°C. Amplified ATACseq libraries were purified using GeneRead Size Selection Kit (Qiagen).
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
ATAC library reads were processed through the ATACseq pipeline (https://github.com/kundajelab/atac_dnase_pipelines). Adapters were removed using Cut-adapt.
Reads were then mapped to hg19 using Bowtie2. Peak calling from BAM files was performed using MACS2 (Zhang et al. 2008).
To create a “masterBED” peak file across conditions, peak files generated for each condition was merged using the merge function from BEDTools (Quinlan and Hall. 2010)
Mean scores from bedGraphs for each individual biological replicate were assigned to masterBED peak files using intersect (default parameters) and merge (-o mean) and used as input for differential analysis using DESeq2 (Love et al. 2014)
All comparisons for NT, TNFα 4 hours and 24 hours treatments were performed using the analysis of deviance function with default parameters in DEseq2. ATACseq peaks with FDR ≤0.001 and fold-change ≥2 or ≤-2 were considered differentially opened or closed
Corresponding biological replicates bedGraphs output from MACS were merged using UCSC BigWig and BigBed tools (Kent et al. 2010)
Genome_build: hg19
Supplementary_files_format_and_content: bigWigs correspond to bedGraphs of narrow peaks output from MACS with a score corresponding to coverage that were merged for all biological replicates; bed corresponds to the peaks present at least in one of the merged biological replicates peak list across all conditions for teloHAEC; narrowPeak corresponds to pooled pseudo replicates and 0.1 IDR thresholded narrowPeaks file for each cell lines across all conditions using the Kundaje lab ATAC-seq pipeline (ENCODE)
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| |
|
| Submission date |
Feb 06, 2019 |
| Last update date |
Jun 25, 2019 |
| Contact name |
Guillaume Lettre |
| E-mail(s) |
Guillaume.Lettre@mhi-humangenetics.org
|
| Organization name |
Montreal Heart Institute
|
| Street address |
5000 Rue Bélanger
|
| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H1T 1C8 |
| Country |
Canada |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE126196 |
Integrative vascular endothelial cell genomics identify AIDA as a coronary artery disease candidate gene (ATACseq) |
| GSE126200 |
Integrative vascular endothelial cell genomics identify AIDA as a coronary artery disease candidate gene |
|
| Relations |
| BioSample |
SAMN10882071 |
| SRA |
SRX5345197 |
