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| Status |
Public on Jun 13, 2018 |
| Title |
Ssrp1_ChIP_seq_rep1,rep2 |
| Sample type |
SRA |
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| Source name |
mESCs
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14 cell line
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| Growth protocol |
The E14 cell line (mESCs) was cultured at 37 °C, 7.5% CO2, on 0.1% gelatin coated plates, in DMEM + GlutaMax™ (Gibco) with 15% fetal bovine serum (Gibco), MEM non- essential amino acids (Gibco), penicillin/streptomycin (Gibco), 550 µM 2-mercaptoethanol (Gibco), and 10 ng/ml of leukaemia inhibitory factor (LIF) (eBioscience).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked with 1% formaldehyde for 20 min followed by quenching for 5 min with the addition of glycine to a final concentration of 0.125 M. After washing with PBS buffer, cells were collected and lysed in Cell Lysis buffer (5 mM Tris pH8.0, 85 mM KCl, 0.5% NP40 ) with proteinase inhibitors (10 µl/mL Phenylmethylsulfonyl fluoride (PMSF), 1 µl/mL Leupeptin and 1 µl/mL Pepstatin). Pellets were spun for 5 min at 5000 rpm at 4°C. Nuclei were lysed in Nuclei Lysis Buffer (1% SDS, 10 mM EDTA, 50 mM Tris HCl) and samples were sonicated for 12 min. Samples were centrifuged for 20 min at 13,000 rpm at 4°C and the supernatant was diluted in IP buffer (0.01% SDS, 1.1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris HCl, 167 mM NaCl) and the appropriate antibody was added and left overnight with rotation at 4°C. Protein A/G Dynabeads (Invitrogen) were added for 1h and after extensive washed samples were eluted in Elution Buffer (1% SDS, 0.1 M NaHCO3). 20 µL of 5 M NaCl were added and samples were reverse-crosslinked at 65°C for 4h. Following phenol-chloroform extraction and ethanol precipitation, DNA was incubated at 37°C for 4h with RNAse (Sigma).
Approximately 10-20 ng of ChIP material was used for library preparation. End-repair and adaptor ligation was prepared as described previously with a few modifications (Tessarz, et al). Double sided size selections (~200 – 650bp) were performed using the MagSI-NGS Dynabeads (MagnaMedics, #MD61021) according to the manufacturer’s instructions. Purified adapter-ligated ChIP material was run on a high sensitivity DNA chip on a 2200 TapeStation (Agilent) to assess size distribution and adaptor contamination.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
ChIP antibody: Anti-Ssrp1 (Biolegends)
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| Data processing |
ChIP-seq files were aligned using Bowtie2 (v 2.2.6). BAM files were converted to bigwig (10 bp bin) and normalised to x1 sequencing depth. Duplicated reads were removed.
Genome_build: mm10
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| Submission date |
Dec 05, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter Tessarz |
| E-mail(s) |
ptessarz@age.mpg.de
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| Organization name |
Max Planck Institute
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| Street address |
Joseph-Stelzman-Str 9b
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| City |
Cologne |
| State/province |
Nordrhein-Westfalen |
| ZIP/Postal code |
50931 |
| Country |
Germany |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE90906 |
Transcriptional repression by FACT is linked to regulation of chromatin accessibility at the promoter of ES cells |
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| Relations |
| BioSample |
SAMN06111832 |
| SRA |
SRX2396606 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2417328_Ssrp1.bw |
319.2 Mb |
(ftp)(http) |
BW |
| GSM2417328_broadenrich.E14_Ssrp1_peaks.tab.gz |
283.7 Kb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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