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| Status |
Public on May 16, 2017 |
| Title |
EC-EA-3702-OCT4_MIT_1 |
| Sample type |
SRA |
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| Source name |
embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6-129
age: E3.5
cell cycle: mitotic
chip antibody: Oct4 (Santa Cruz SC-8628)
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| Treatment protocol |
Mitotic arrest was done as follows: 80-90% confluent mouse ESCs were split 1:3 on gelatinized plates the day before synchronization. Nocodazole at 100ug/ml was added in the medium for 7hr prior to collection by mitotic shake-off.
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| Growth protocol |
V6.5 ESCs were cultured on irradiated feeder cells in KO-DMEM (Invitrogen) supplemented with L-Glutamine, penicillin-streptomycin, nonessential amino acids, β-mercaptoethanol, 1000 U/ml LIF and 15% heat-inactivated fetal bovine serum (FBS). Prior to synchronization, cells were expanded on gelatinized plates in the presence of 2i, MEK1 inhibitor (PD98059) and Gsk3 inhibitor (Chir99021) for at least 2 passages.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Asynchronous or mitotic ESCs were crosslinked in 1% formaldehyde at room temperature (RT) for 10 minutes and quenched with 125mM glycine 5 mins at RT. 25-50M ESCs were used for the TF ChIPs and 10M for the H3K27acetylation ChIP. Cell pellets were washed twice in PBS and resuspended in 400ul lysis buffer per 20 million cells (10mM Tris pH8, 1mM EDTA, 0.5% SDS). Cells were sonicated in a bioruptor device (30 cycles 30sec on/off, high setting) and spin down 10 minutes at 4 degrees at maximum speed. Supernatants were diluted 5 times with dilution buffer (0.01%SDS, 1.1% triton,1.2mM EDTA,16.7mM Tris pH8, 167mM NaCl) and incubated with the respective antibody (2-3ug/10M cells) O/N with rotation at 4oC. Next day, protein G Dynabeads (ThermoScientific) preblocked with BSA protein (100ng per 10ul Dynabeads) were added (10ul blocked Dynabeads per 10 million cells) and incubated for 2-3 hours at 4oC. Beads were immobilized on magnet and washed twice in low salt buffer (0.1% SDS,1% triton, 2mM EDTA, 150mM NaCl, 20mM Tris pH8), twice in high salt buffer (0.1% SDS,1% triton, 2mM EDTA, 500mM NaCl, 20mM Tris pH8), twice in LiCl buffer (0.25M LiCl, 1% NP40, 1% deoxycholic acid (sodium salt), 1mM EDTA, 10mM Tris pH8) and once in TE. DNA was then eluted from the beads by incubating with 150ul elution buffer (1% SDS, 100mM NaHCO3) for 20 minutes at 65 degrees (vortex every 10min). Supernatants were collected and reverse-crosslinked by incubation at 65 degrees O/N in presence of proteinase K. After Rnase A treatment for 1hr at 37oC, DNA was purified using the Qiagen minElute kit. 6-10ng of immunoprecipitated material was used for ChIP-seq.
ChIP-seq library preparation using the KAPA Hyper prep kit from KAPA Biosystems. Libraries were sequenced on an Illumina HiSeq 5000 platform on the SE50 mode.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Reads were trimmed for residual adapters (cutadapt version 1.8.1).
Alignment to the mouse reference genome version mm10 was performed using standard parameters, permitting maximum one mismatch in seed alignment (Bowtie version 2.2.5).
Reads marked as positional duplicates or falling into mouseENCODE blacklisted genomic regions (liftOver to mm10; Dunham et al. 2012) were filtered out.
Coverage tracks in RPKM were generated from alignments using bamCoverage (V2.3.6) at the bin size of 10.
ChIP-seq peaks (enrichment of signals over background) were called at 10-5 (MACS version 2.1.1), and peaks detected in more than half of biological replicates were retained for further analysis.
Genome_build: mm10
Supplementary_files_format_and_content: BigWig files for coverage tracks; narrowPeak files for peak coordinates.
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| Submission date |
Dec 22, 2016 |
| Last update date |
Oct 10, 2023 |
| Contact name |
Effie Apostolou |
| E-mail(s) |
efa2001@med.cornell.edu
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| Organization name |
Weill Cornell Medicine
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| Street address |
1161 York Avenue, Apt 8A
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE92846 |
Stem cell regulatory elements remain bookmarked by selected transcription factors and epigenetic marks during mitosis |
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| Relations |
| BioSample |
SAMN06176553 |
| SRA |
SRX2441340 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2438471_EC-EA-3702-OCT4_MIT_1.bw |
221.1 Mb |
(ftp)(http) |
BW |
| GSM2438471_EC-EA-3702-OCT4_MIT_1.narrowPeak.gz |
23.9 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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