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| Status |
Public on Sep 15, 2017 |
| Title |
ARNT_ChIPSeq |
| Sample type |
SRA |
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| Source name |
HUVEC cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HUVEC
chip antibody: ARNT (Novus, NB-100-110)
passagges: from 4 to 6
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| Treatment protocol |
Cells were exposed 16h to hypoxia in a 1% O2, 5% CO2, 94% N2 gas mixture in a Whitley Hypoxystation H35 (Don Withley Scientific).
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| Growth protocol |
Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from Lonza (Lonza, C2519A) and grown in Endothelial Medium Bullet Kit (EGM‐2, Lonza, CC‐3162).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
HUVEC cells fixed and protein‐DNA complexes crosslinked by treatment with 1% formaldehyde, and then quenched with glycine (125mM). Next, cells were washed 3 times with ice‐cold PBS, collected using a cell scraper and resuspended in ice‐cold lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris‐HCl pH 8.1). DNA was sheared by using a sonicator (Diagenode Bioruptor, Belgium). Samples were diluted 1/10 with dilution Buffer (0.01% SDS, 1.1% Triton X‐100, 1.2 mM EDTA, 16.7 mM Tris‐HCl pH 8.1, 167 mM NaCl) and then precleared with Salmon Sperm DNA/Protein A agarose 50% slurry (Fast Flow, Millipore, 16‐156.) Sample were immunoprecipitated with different antibodies at 4°C rocking overnight; HIF1A (PM14), EPAS1 (PM9), [57] ARNT (Novus NB‐100‐110) and pre‐immune serum used as negative control.Immunocomplexes were recovered by addition of Salmon Sperm DNA/Protein A agarose 50% slurry. Then samples were sequentially washed in Low Salt Wash Buffer (0.1% SDS, 1% Triton X‐100, 2 mM EDTA, 20 mM Tris‐HCl pH 8.1, 150 mM NaCl), High Salt Wash Buffer (0.1% SDS, 1% Triton X‐100, 2 mM EDTA, 20 mM Tris‐HCl pH 8.1, 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1% NP‐40, 1% NaDesoxycholate, 1 mM EDTA, 10 mM Tris‐HCl pH 8.1), and twice in TE buffer (10 mM Tris‐HCl pH 8, 1 mM EDTA). Elution of protein‐bound DNA was performed using 500 μl of fresh elution buffer (1% SDS, 0.1 M NaHCO3). Next crosslinking of all samples was reversed by the overnight incubation with 200 mM NaCl at 65°C. Next day, proteins were removed by the addition of 10 μl of 0.5 M EDTA, 20 μl Tris‐HCl pH 6.5 and proteinase K (40 μg/sample). Then immunoprecipitated DNA was purified by Phenol:Chloroform:Isoamyl alcohol extraction and ethanol precipitation (UltraPure™ phenol:Chloroform:Isoamyl Alcohol 25:24:1, Invitrogen, 15593‐031).
Libraries were prepared according to Illumina ChIP‐Seq kit's intructions (Illumina, IP‐202‐1012).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Illumina adaptor sequences were trimmed from the ChIP-seq reads using Trimgalore and the reads were then aligned to Genome Reference Consortium GRCh37 (hg19) using BWA ChIP-seq: Low quality mapping was removed (MapQ < 15) using SAMtools
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: wig files were generated using genomeCoverageBed (v2.25.0) and wigToBigWig tools.
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| Submission date |
Nov 14, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Luis del Peso |
| E-mail(s) |
luis.peso@uam.es
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| Phone |
+34 91 585 4440
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| Organization name |
Universidad Autonoma de Madrid
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| Department |
Biochemistry
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| Lab |
IIB-252
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| Street address |
Arzobispo Morcillo
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| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE89836 |
Genome-wide mapping of HIF binding sites by ChIP-seq in HUVEC cells exposed to hypoxia |
| GSE89841 |
Transcriptional repression in hypoxia is mediated by the Sin3A histone deacetylase complex |
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| Relations |
| BioSample |
SAMN06015692 |
| SRA |
SRX2346893 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2390643_ARNT_ChIPSeq.bw |
78.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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