|
| Status |
Public on Nov 01, 2016 |
| Title |
H3K27me3 normoxia |
| Sample type |
SRA |
| |
|
| Source name |
MCF7
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
oxygen state: normoxia
timepoint: 0 hours
oxygen concentration: 21%
chip antibody: anti-H3K27me3 (Upstate, catalog# 07-449, lot# DAM1514011)
|
| Treatment protocol |
For hypoxic exposure, cells were transferred to a MACS VA500 microaerophilic workstation (Don Whitley Scientific, Shipley, UK) for the indicated duration. The atmosphere in the chamber consisted of <0.02% O2, 5% H2, 5% CO2, and 74% N2.
|
| Growth protocol |
MCF7 (human mammary adenocarcinoma) were cultured at 37°C, 5% CO2, 100% humidity in Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12 (DMEM/F12 1:1; MCF7) and DMEM McCoy’s 5A medium (DU145). The culture medium was supplemented with 10% fetal calf serum (FCS), 200 mM L-glutamine and antibiotics.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
MCF7 cells were transferred to hypoxic culturing conditions for the indicated durations and immediately fixed to avoid reoxygenation. After 24 hours of hypoxia, cells were reoxygenated for 8 hours. Cells were disrupted by sonication, yielding genomic DNA fragments ranging from 200-1000 bp, with a bulk size of 200-500bp. For each immunoprecipitation 10-20 million cells were used. ChIPs were performed and analyzed using standard protocols (antibody: H3K4me3 and H3K27me3 (07-449; Upstate)).
ChIP-seq libraries were prepared for sequencing using standard 36 bp paired-end protocol for the Illumina Genome Analyser IIx (GAIIx). Two lanes were used per sample for sufficient coverage.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
Sample name: 5
|
| Data processing |
Image processing and base calling was performed using Illumina software tools provided by the manufacturer.
Subsequent paired-end genome alignment was performed using Novoalign with Human Genome 18 (HG18) used as a reference genome. Only uniquely aligned reads were used for further analysis.
To remove PCR artifacts all data were collapsed prior to peak calling.
To identify H3K4me3 and H3K27me3 marked regions the peak caller Findpeaks (version 4.0) was used using standard settings.
Genome_build: HG18
Supplementary_files_format_and_content: WIG files containing H3K4me3 and H3K27me3 marked regions
|
| |
|
| Submission date |
Jul 17, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Michiel Adriaens |
| E-mail(s) |
michiel.adriaens@maastrichtuniversity.nl
|
| Organization name |
Maastricht University
|
| Department |
Maastricht Centre for Systems Biology - MaCSBio
|
| Street address |
Universiteitssingel 60
|
| City |
Maastricht |
| ZIP/Postal code |
6229 ER |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL10999 |
| Series (2) |
| GSE71030 |
H3K4me3 and H3K27me3 ChIP-seq profiling in MCF7 cell lines under hypoxia and reoxygenation |
| GSE71031 |
Hypoxia increases genome-wide bivalent epigenetic marking by specific gain of H3K27me3 |
|
| Relations |
| BioSample |
SAMN03876576 |
| SRA |
SRX1099916 |
