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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 29, 2023 |
| Title |
ATACseq_ESC_dTrim24_DegrAct_12h_r1 |
| Sample type |
SRA |
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| Source name |
embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic stem cells
genotype: degron-tagged Trim24
strain: 129S6/SvEvTac
treatment condition: degraded Trim24; activated p53 - 12 hour
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| Treatment protocol |
Cells (0.25x10^6) were seeded into 6-well plates the day before the experiment. Doxorubicin (1uM) or dTAG13 (500nM) compound were added to specific samples to activate p53 or degrade degron tagged alleles. For activation of p53 via alternative pathways, cells were treated with 20 µM nutlin3a or cells were subjected to 60Gy ionizing radiation and left for 4 hours. ATAC-seq was performed according to the previously described protocol(Corces, M. R. et al. Nat. Methods, 2017) with modifications. Briefly, 5x10^4 cells were resuspended in 1 ml of cold ATAC-seq resuspension buffer (RSB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2 in water). Cells were centrifuged at 500g for 5 min in a pre-chilled (4°C). Cell pellets were suspended in 50 μl of ATAC-seq RSB containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin by pipetting up and down three times. This cell lysis reaction was incubated on ice for 3 min. After lysis, 1 ml of ATAC-seq RSB containing 0.1% Tween-20 (without NP40 or digitonin) was added, and the tubes were inverted to mix. Nuclei were then centrifuged for 10 min at 500 g (4°C). Nuclei were suspended in 50 μl of Tn5 transposition mix (25 μl 2× TD buffer, 2.5 μl transposase (100 nM final), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, and 5 μl water) by pipetting up and down six times. Transposition reactions were incubated at 37°C for 30 min in a thermomixer with shaking at 1,000 rpm. Reactions were cleaned up using the MinElute PCR Purification Kit (Qiagen). The eluted transposed DNA was subjected to PCR amplification using Q5 High-Fidelity Polymerase (NEB) for 7 cycles.
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| Growth protocol |
Mouse ES cells were cultured as previously described (Mohn, F. et al. Mol. Cell, 2008). Briefly, cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen), supplemented with 15 % Fetal Calf Serum (Invitrogen), L-Glutamine (Gibco) and nonessential amino acids (Gibco), betamercaptoethanol (Sigma) and leukemia inhibitory factor (LIF; produced in-house). All experiments were performed with cells grown for several passages on plates coated with 0.2% gelatin (Sigma).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was purified using the MinElute PCR Purification Kit (Qiagen).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Illumina RTA 2.4.11 and bcl2fastq2 v2.17 was used for basecalling and demultiplexing. ATAC-seq reads were trimmed using cutadapt version 1.18, (Martin, M. EMBnet.journal 17, 10 (2011)) with parameters -a CTGTCTCTTATACACA -A CTGTCTCTTATACACA -m 5 --overlap=1 and then mapped to mm10 using QuasR (Gaidatzis et al, Bioinformatics 2015) with default parameters.
Supplementary files format and content: wig files were generated using the qExportWig function from the QuasR package with scaling=1e6 and pairedAsSingle=TRUE . The scores in the wig file therefore represent the number of 5'-read ends (Tn5 insertion sites) per 100bp window and per million reads in the library.
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| Submission date |
Apr 11, 2022 |
| Last update date |
May 01, 2023 |
| Contact name |
Luke Thomas Isbel |
| E-mail(s) |
luke.isbel@adelaide.edu.au
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| Organization name |
SAiGENCI
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| Lab |
Isbel
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| Street address |
4 North Terrace, AHMS, Lvl 9
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| City |
Adelaide |
| State/province |
South Australia |
| ZIP/Postal code |
5000 |
| Country |
Australia |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE200582 |
Chromatin opening by p53 is confined by Trim24 in a histone methylation-dependent manner [ATAC-seq] |
| GSE200586 |
Chromatin opening by p53 is confined by Trim24 in a histone methylation-dependent manner |
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| Relations |
| BioSample |
SAMN27517578 |
| SRA |
SRX14812314 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6038184_ATACseq_ESC_dTrim24_DegrAct_12h_r1.wig.gz |
10.6 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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