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| Status |
Public on Jun 25, 2019 |
| Title |
H3K27ac_teloHAEC_24h_r2 |
| Sample type |
SRA |
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| Source name |
Endothelial Cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: immortalized Human Aortic Endothelial Cells
passages: passages 23 to 25
chip antibody: H3K27ac (Diagenode; C15410196)
tnf-alpha treatment: 24h
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| Treatment protocol |
Endothelial cells were treated with TNF-alpha prepared in culture media at 10 ng/mL for 4h and 24h periods
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| 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 g/mL of streptomycin. TeloHAEC (below 3 passages) were maintained under a 5% CO2 atmosphere at 37 C. Endothelial cells were seeded at 1.4x10^5 cells per 100 mm plates(1 plate per condition, 3 independent biological replicates), grown to 90-100% confluency (refreshed media every 2-3 days) and subjected to TNF-alpha treatment.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were washed with HBSS Gibco and fixed in paraformaldehyde (PFA) 1% for 10 minutes at room temperature (RT). PFA was quenched in 134 mM glycine for 5 minutes at RT. Fixed cells were washed with ice-cold PBS and collected with a cell scraper in ice-cold PBS. Cells were pelleted by centrifugation, washed in ice-cold PBS, and pelleted again before snap freezing in liquid nitrogen. Fixed cells were subject to lysis in 5 mM PIPES-pH 8.5, 85 mM KCl, 1% (v/v) IGEPAL CA-630, 50 mM NaF, 1 mM PMSF, 1 mM Phenylarsine Oxide, 5 mM Sodium Orthovanadate and protease inhibitor cocktail. Nuclei were then lysed in 50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% (w/v) SDS, 50 mM NaF, 1 mM PMSF, 1 mM Phenylarsine Oxide, 5 mM Sodium Orthovanadate and protease inhibitor cocktail. Lysed nuclei were sonicated to yield fragments within a 100 to 500 bp range. Chromatin immunoprecipitation was performed using 3.7 µg of H3K27ac antibody (Diagenode) per samples containing 5x10^5 cells (no antibody for input). Samples were purified through silica-membrane-based columns prior to library preparation.
Library construction was performed using KAPA Hyper Prep Kit (Kapa Biosystems) for Illumina using with adapters from TruSeq DNA LT Sample Prep Kit (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
H3K27ac ChIPseq library raw reads were filtered for quality (phred33≥30) and length (n≥32) and adapter sequences were removed using Trimmomatic (Bolger et al. 2014)
Filtered reads were aligned to hg19 using BWA and peaks subsequently called using MACS2 (Zhang et al. 2008) with non-IP input DNA as control
Corresponding bedGraphs output of biological replicates and input controls from MACS were merged using UCSC BigWig and BigBed tools (Kent et al. 2010)
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)
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; input bigWigs corresponds to MACS2 output for input controls for each condition; bed corresponds to the peaks present at least in one of the merged biological replicates peak list across all conditions; narrowPeak corresponds to MACS2 output of narrowPeaks format for each biological replicates across all conditions
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| Submission date |
Feb 06, 2019 |
| Last update date |
Jun 25, 2019 |
| Contact name |
Guillaume Lettre |
| E-mail(s) |
[email protected]
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| Organization name |
Montreal Heart Institute
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| Street address |
5000 Rue Bélanger
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| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H1T 1C8 |
| Country |
Canada |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE126197 |
Integrative vascular endothelial cell genomics identify AIDA as a coronary artery disease candidate gene (ChIPseq) |
| GSE126200 |
Integrative vascular endothelial cell genomics identify AIDA as a coronary artery disease candidate gene |
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| Relations |
| BioSample |
SAMN10882055 |
| SRA |
SRX5345181 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3593239_H3K27ac_teloHAEC_24h_rep2.narrowPeak.gz |
2.5 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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