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| Status |
Public on Sep 01, 2016 |
| Title |
hmDIPseq MCF7 hypoxia exp 3 |
| Sample type |
SRA |
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| Source name |
MCF7 cells
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| Organism |
Homo sapiens |
| Characteristics |
oxygen concentration: 0.5% oxygen
dip antibody: anti 5hmC (Active Motif cat 39791)
experiment: 3
replicate: A
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| Treatment protocol |
cells were grown for 24 hours at normoxic or hypoxic oxygen tensions
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| Growth protocol |
MCF7cells were cultured at 37°C in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS), 5ml of 100 U/ml Penicillin-Streptomycin (Pen Strep, Life Technologies) and 5ml of L-Glutamine 200mM
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nucleic acids were subsequently extracted using the Wizard Genomic DNA Purification (Promega, Leiden, The Netherlands) kit according to instructions, with all buffers supplemented with DFO (200 µM), dissolved in 80 µL PBS-DFO with RNAse A (200 units, NEB, Ipswich, MA, USA), incubated for 10 minutes at 37°C. After proteinase K addition (200 units) and incubation for 30 minutes at 56°C, DNA was purified using the QIAQuick blood and tissue kit (all buffers supplemented with DFO), eluted in 100 µL of a 10 mM Tris, 1mM EDTA solution (pH 8) and stored at -80°C until further processing.
Library preparations and DNA immunoprecipitations were as described by Taiwo and colleagues (Nat Protoc 2012), using established antibodies targeting 5mC and 5hmC.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
raw read counts input and hmDIP.txt
differential hydroxymethylation
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| Data processing |
alignment was done using Bowtie 0.12.8 (-v 2 -m 1 --best -S), or BisMark
SeqMonk 0.29.0 was used to determine read counts at coding gene promoters (2000 bp upstream to 500 bp downstream of the gene start site) as described in ensembl release grch37.
differential methylation or hydroxymethylation was determined using EdgeR version 3.6.8, correcting for experiment variable, and this for pairwise comparisons between experiments and comparisons within single experiments. for hmDIPseq, library sizes were corrected to reflect the ratio at input
Genome_build: Hg19
Supplementary_files_format_and_content: raw read .txt files contain the numer of reads in each experiment at the promoter. These are compared using EdgeR, generating result files with logCPM (log2 counts per million), logFC (log fold change) and P-values of change.
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| Submission date |
Apr 08, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Bernard thienpont |
| Organization name |
VIB
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| Department |
Vesalius Research Center
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| Lab |
Translational genetics
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| Street address |
Herestraat 49
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| City |
Heverlee |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE71400 |
Tumor hypoxia causes DNA hypermethylation by reducing TET activity (DIP-Seq) |
| GSE71403 |
Tumor hypoxia causes DNA hypermethylation by reducing TET activity |
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| Relations |
| BioSample |
SAMN04625740 |
| SRA |
SRX1688421 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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