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| Status |
Public on Feb 20, 2024 |
| Title |
Input-2 |
| Sample type |
SRA |
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| Source name |
E14
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| Organism |
Mus musculus |
| Characteristics |
developmental stage: E14
cell type: embryonic stem cells
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| Growth protocol |
E14 mouse embryonic stem cells (mESCs) were cultured with mouse ESC medium (DMEM high glucose supplemented with 15% defined FBS (Hyclone), 2mM L-glutamine (Gibco), 1X PenStrep (Gibco), 100μM MEM non-essential amino acids (Gibco), 100μM β-mercaptoethanol (Gibco) and 1000 U/mL leukemia inhibitory factor (LIF; ESGRO, Millipore)). All the EC and ESC cell lines used in this study were cultured on gelatin-coated tissue culture plates and all the cell lines were cultured at 37°C with 5% CO 2 .
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
10 million cells were cross-linked with 1% formaldehyde for 10 min at room temperature, with the formaldehyde then quenched by the addition of 200 mM glycine and cells then washed once with ice-cold PBS. Cell lysis was carried out with a buffer containing 10 mM Tris-Cl (pH 8), 100 mM NaCl, 10 mM EDTA, 0.25% Triton X-100 and protease inhibitor cocktail (Roche). Cells were then resuspended in 1% SDS lysis buffer containing 50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 1% SDS, 2 mM EDTA, 1% Triton X-100, 0.1% NaDOC and protease inhibitor cocktail. The suspension was nutated for 15 mins at 4°C before spinning down to collect the chromatin pellet. The pellet was then washed twice with 0.1% SDS lysis buffer containing 50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 0.1% SDS, 2 mM EDTA, 1% Triton X-100, 0.1% NaDOC, 1 mM PMSF and protease inhibitor cocktail. Sonication was conducted using the Branson sonifier 250 on ice (11 cycles, power amplitude of 35%, 30s pulses on with 59.9 s pulses off). The chromatin solution was clarified by centrifugation at 20,000 g at 4°C for 45 mins and then pre-cleared with Dynabeads protein A (Life Technologies) for 2 hrs. The pre-cleared chromatin sample was incubated with 50 μl of Dynabeads protein A loaded with 5 μg antibody overnight. The beads were then washed three times with 0.1% SDS lysis buffer, once with 0.1% SDS lysis buffer/0.35 M NaCl, once with 10 mM Tris-Cl (pH 8), 1 mM EDTA, 0.5% NP40, 0.25 LiCl, 0.5% NaDOC, and once with TE buffer (PH8.0). The immunoprecipitated material was eluted from the beads by heating for 45 min at 68°C in 50 mM Tris-Cl (pH 7.5), 10 mM EDTA, 1% SDS. To reverse the crosslinks, samples were incubated with 1.5 μg/ml Pronase at 42°C for 2 hr followed by 67°C for 6 hr. The samples were then extracted with phenol chloroform isoamyl alcohol followed by chloroform, ethanol precipitated in the presence of glycogen, and re-suspended in TE buffer.
The ChIP-Seq libraries were prepared using Illumina TruSeq ChIP Sample Prep Kit (Catalog #: IP-202-1012) as per manufacturer's instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Libraries were mapped to mm9 genome using STAR with the options –alignIntronMax and –AlignEndsType set to 1 and EndToEnd respectively.
peaks were called using MACS2 and bigiwg files were produced by deeptools, bamCoverage --normalizeUsing RPGC
Genome_build: mm9
Supplementary_files_format_and_content: narrowPeaks indicating the binding region of the libraries on the Genome
Supplementary_files_format_and_content: bigwig file, indicating the enrichment of the libraries on the Genome
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| Submission date |
Feb 21, 2021 |
| Last update date |
Feb 20, 2024 |
| Contact name |
Yingying Zeng |
| E-mail(s) |
[email protected]
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| Organization name |
Institute of Molecular and Cell Biology
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| Lab |
Epigenetics and Cell Fates Laboratory
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| Street address |
61 Biopolis Drive
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| City |
Singapore |
| State/province |
--- Select a state --- |
| ZIP/Postal code |
138673 |
| Country |
Singapore |
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| Platform ID |
GPL21103 |
| Series (2) |
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| Relations |
| BioSample |
SAMN18022153 |
| SRA |
SRX10145162 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5098623_4_Ssrp1-2_Input.bw |
170.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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