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| Status |
Public on Oct 28, 2021 |
| Title |
H3K9me3_input |
| Sample type |
SRA |
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| Source name |
mESC
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| Organism |
Mus musculus |
| Characteristics |
cell type: mESC
strain: FVB/NJ
genotype: not provided
chip antibody: none (input)
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| Growth protocol |
All mESC lines were cultured in serum conditions (Knockout DMEM (10829-018; Gibco), 10% FBS (DE14-801F; BioWhittaker), NEAA (11140; Gibco), L-Glutamine (25030-123; Gibco), Sodium Pyruvate (11360; Gibco), 2-Mercaptoethanol (31350; Gibco) and Leukemia Inhibitory Factor (ESG1107; Millipore)) plus MEK inhibitor PD0325901 (1 mM) and GSK3 inhibitor CHIR99021 (3 mM) (Axon Medchem) on 0.1% gelatin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were cross linked with 1% formaldehyde (M134-200ML; VWR) for 8 min at room temperature and glycine (125 mM; G8790-1KG; Sigma) was used to quench cross-linking for 5 min. Cells were washed twice with cold PBS and lysed in NP Buffer (150 mM NaCl, 50 mM Tris–HCl (pH 7.5), 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, Protease Inhibitor Cocktail (05056489001; Roche)). Nuclei were sheared by sonication (Covaris). For each H3K9me3 ChIP-seq experiment, 25 µg of sample chromatin was mixed with 50 ng spike-in Drosophila chromatin (53083; Active Motif). Mixture of experimental chromatin and spike-in chromatin was then incubated with a mix containing 4 µg of H3K9me3 antibody (abcam ab8898) and 2 µg of spike-in antibody (104597; Active Motif) at 4°C overnight. The next day, Protein A Sepharose beads (175280-01; GE Health Care) were first blocked with 1mg/ml BSA (10484; Affymetrix) and then added to each chromatin-antibody mix and incubated at 4°C for at least 3 h. After immunoprecipitation, beads were washed with low-salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl (pH 8.1), 150 mM NaCl), high-salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl (pH 8.1), 500 mM NaCl), LiCl washing buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris–HCl (pH 8.1)) and TE buffer (10 mM Tris–HCl (pH 8.0), 1 mM EDTA). DNA was extracted using phenol-chloroform-isoamylol (15593-049; Life Technologies)
About 20ng of DNA from mESCcells was used for library preparation with the KAPA Hyper Kit following manufacturer's instructions and sequenced at Macrogen on HiseqX
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
HiSeq X Ten |
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| Data processing |
Quality assessment of the raw sequencing reads was done using FastQC v0.11.2. Adapters were removed by TrimGalore v0.4.5 using default parameters for paired-end Illumina reads, after which, quality filtering was performed by the same software. Reads smaller than 20bps and those with an error rate (TrimGalore option "-e") higher than 0.1 were discarded, after which a final quality assessment of the filtered reads was done with FastQC to identify possible biases left after filtering
The remaining reads were mapped to the mouse reference genome (build mm10) using the Bowtie2 v2.3.5 using default parameters with the following exceptions: “–very-sensitive".
The peak calling was done with SICER2
Genome_build: mm10
Supplementary_files_format_and_content: bigwig and peaks
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| Submission date |
May 05, 2021 |
| Last update date |
Jan 12, 2023 |
| Contact name |
Lucia Daxinger |
| E-mail(s) |
[email protected]
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| Organization name |
Leiden Univeristy Medica Center
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| Street address |
Albinusdreef 2
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| City |
Leiden |
| ZIP/Postal code |
2333ZA |
| Country |
Netherlands |
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| Platform ID |
GPL21273 |
| Series (2) |
| GSE173912 |
Morc3 silences endogenous retroviruses in mouse embryonic stem cells [H3K9me3 ChIP-seq] |
| GSE173917 |
Morc3 silences endogenous retroviruses in mouse embryonic stem cells |
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| Relations |
| BioSample |
SAMN19022925 |
| SRA |
SRX10783413 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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