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| Status |
Public on Oct 06, 2021 |
| Title |
16_time60_2SP-POL2 |
| Sample type |
SRA |
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| Source name |
HUVEC
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| Organism |
Homo sapiens |
| Characteristics |
cell type: HUVEC
antibody used for chip: Ser2-p-RNA polymerase II
treatment: VEGF-treatment
time: time60
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| Treatment protocol |
80-90% confluent HUVECs plates were washed with PBS once and then serum starved in EBM-2 Basal Medium containing 0.5% FBS for 16 hrs. HUVECs were stimulated with or without 50 ng/ml VEGF-a (Peprotech, Rocky Hill, NJ) for indicated periods thereafter.
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| Growth protocol |
HUVECs were purchased from Lonza Japan Ltd. (Tokyo, Japan) and cultured and passaged every 2 or 3 days in EBM-2 complete medium composed of EBM-2 Basal Medium (Lonza) supplemented with EGM-2 SingleQuot Kit Suppl. & Growth Factors (Lonza) and 3% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA). We sub-cultured HUVECs when they reached 80 to 90% confluence to avoid excessive floaters. HUVECs were used within passage 4 to 7 for all experiments in this work. All cells were cultured at 37 ºC and 5% CO2 in a humidified incubator.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation (ChIP) was performed as previously described (Kanki et al., 2011; Kanki et al., 2017). In brief, 10 million HUVECs were cross-linked with 1% formaldehyde (Wako) for 10 min. For p65 and FLAG ChIP analysis, cells were fixed with 2 mM EGS (ethylene glycol bis [succinimidyl succinate]) (Thermo Fisher Scientific) for 1 hr prior to formaldehyde fixation. After neutralization by using 0.2 M glycine, cells were collected, re-suspended in 2 ml SDS lysis buffer composed of 10 mM Tris-HCl; pH8.0 (Thermo Fisher Scientific), 150 mM NaCl (Thermo Fisher Scientific), 1% SDS (Sigma-Aldrich), 1mM EDTA; pH 8.0 (Thermo Fisher Scientific), cOmplete™ EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich), and fragmented by the Picoruptor (40 cycles for histones and p65, and 20 cycles for FLAG, 30 sec on/30 sec off; Diagenode, Liege Science Park, Belgium). Sonicated solution was diluted by ChIP dilution buffer (20 mM Tris-HCl; pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 (Sigma-Aldrich)) up to 10.3 ml, which was used for immunoprecipitation (10 ml) and the rest 300 μl as a non-immunoprecipitated chromatin (INPUT). Specific antibody was bound with magnetic Dynabeads M-280 and applied to diluted sonicated solution for immunoprecipitation. Antibodies against H3K4me3, H3K27ac, H3K9me2, H3K27me3 (MAB Institute, Inc. Hokkaido, Japan), p65 (abcam for ChIP-Seq and Santa Cruz Biotechnology Inc for ChIP-PCR), and FLAG (Sigma-Aldrich) were used. Prepared DNA was quantified using NanoDrop 2000 (Thermo Fisher Scientific) and more than 10 ng of DNA was processed for qPCR or sequence.
Libraries were prepared according to Illumina's instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
ChIP-seq data of human endothelial cells through VEGF-induced angiogenic signaling
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| Data processing |
Illumina CASAVA1.8.2 software used for basecalling and demultiplexing.
Reads were trimmed with Trimmomatic.
The trimmed reads were aligned to the mouse reference genome hg19 with Bowtie2.
Sequencing tracks normalized by the mapped read counts were generated with deepTools.
Genome_build: Genome Reference Consortium Human Genome 19 (hg19)
Supplementary_files_format_and_content: bigwig
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| Submission date |
Jan 19, 2021 |
| Last update date |
Oct 06, 2021 |
| Contact name |
Yasuharu Kanki |
| E-mail(s) |
kanki@lsbm.org
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| Organization name |
The University of Tokyo
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| Street address |
4-6-1 Komaba, Meguro-ku
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| City |
Tokyo |
| ZIP/Postal code |
153-8904 |
| Country |
Japan |
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| Platform ID |
GPL10999 |
| Series (1) |
| GSE159075 |
Histone modification profiles on Human Umbilical Vein Endothelial Cells (HUVECs) under Vasucular Endothelial Cell Growth Factor (VEGF) stimulation |
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| Relations |
| BioSample |
SAMN17383708 |
| SRA |
SRX9896198 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5026281_16_time60_2SP-POL2.fastq.bigWig |
190.0 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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