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| Status |
Public on Oct 28, 2021 |
| Title |
Morc3_input_B |
| Sample type |
SRA |
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| Source name |
mESC
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| Organism |
Mus musculus |
| Characteristics |
cell type: mESC
strain: V6.5
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 inhibi- tor 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 ChIP Buffer 1 (10mM Tris HCl (pH 8.0), 0,25% Triton X-100, 10mM EDTA, 0.5mM EGTA, Protease Inhibitor Cocktail (05056489001; Roche) and 1mM PMSF) for 15 minutes on a rotator at 4C. After centrifugation 5 minutes at 1,400g at 4C, cells were resuspended in ChIP Buffer 2 (10mM Tris-HCl (pH 8.0), 200mM NaCl, 10mM EDTA, 0.5mM EGTA, Protease Inhibitor Cocktail (05056489001; Roche) and 1mM PMSF). After centrifugation 5 minutes at 1,400g at 4C, cells were resuspended in ChIP Buffer 3 (10mM Tris-HCl (pH 8.0), 10mM EDTA, 0.5mM EGTA, 0,1% SDS, Protease Inhibitor Cocktail (05056489001; Roche) and 1mM PMSF and sonicated with Covaris S2 (Duty cycle: 10%, Intensity:5, Cycle/Burst:200 to obtain fragment between 200 and 600bp. Sheared chromatin was centrifuged at 12,000 g for 10 min at 4°C to discard the pellets. The supernatant was then diluted 10 times with ChIP dilution buffer (16.7mM Tris-HCl (pH8), 0.01% SDS, 1.1% TritonX-100, 1.2mM EDTA, 167mM NaCl) and incubated overnight with 5 g Morc3 antibody (Rockland; 100-401-N96S) or Rabbit IgG (Abcam ab37415). An input fraction for each sample was retained for downstream analysis. Samples were then incubated with Dynabeads Protein A (Invitrogen 10001D) for at least 4h rotating at 4C. 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). IP DNA and Input DNA samples were extracted with phenol-chloroform-isoamylalcohol (15593049; Fisher Scientific).
DNA from mESc was used for library preparation with the KAPA Hyper prep kit following manufacturer's instructions and libraries were sequenced on Illumina HiSeq 2000.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Sequencing adapters were remove with Trimmomatic (0.38) and read were mapped to the reference genome (mm10) using bowtie2 (2.3.4) with parameter "--very-sensitive-local"
Peaks were called using MACS2 (2.1.1) with the following parameters ( -g mm). Input dataset containing whole genomic DNA fragments were used as control.
Genome browser tracks (bigwig) were generated using Deeptools (3.1.2) with parameters "--effectiveGenomeSize 2652783500 --binSize 10 --smoothLength 50 --normalizeUsing RPKM"
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 |
GPL13112 |
| Series (2) |
| GSE173913 |
Morc3 silences endogenous retroviruses in mouse embryonic stem cells [Morc3 ChIP-seq] |
| GSE173917 |
Morc3 silences endogenous retroviruses in mouse embryonic stem cells |
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| Relations |
| BioSample |
SAMN19022982 |
| SRA |
SRX10783419 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5282857_Morc3_input_B.bw |
599.5 Mb |
(ftp)(http) |
BW |
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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