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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 11, 2013 |
| Title |
SMAD1_36h_PV |
| Sample type |
SRA |
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| Source name |
EpiLC
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
chip antibody: SMAD1(BMR00479)
genotype: Prdm14-mVenus reporter (PV)
cell type: EpiLC
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| Growth protocol |
ESCs were induced into EpiLCs as described previously (Hayashi et. al., 2011). 1×105 ESCs were cultured in a well of a 12-well plate coated with human plasma fibronectin (Merck Millipore) (16.7 mg/ml) in N2B27 medium containing Activin A (20 ng/ml; Peprotech), bFGF (12 ng/ml; Invitrogen), and KSR (1 %). Medium was changed 24 hrs later, and the cells were cultured for another 24 hrs. The resultant EpiLCs were then cultured under a floating condition by plating 2 x 103 cells in a well of a lipidure coated U-bottom 96-well plate (Thermo Scientific, Waltham, MA) in a GK15+LIF medium in the presence or absence of the cytokines [BMP4 (500 ng/ml), WNT3A (50 ng/ml)].
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were trypsinized, washed, and collected by centrifugation. For crosslinking, the pellet was resuspended in PBS containing 1% formaldehyde, incubated for 10 min, and quenched with 125 mM glycine. The fixed cells were resuspended in 1 ml of cold LB1 (20 mM Tris, 10 mM NaCl, 2.5 mM MgCl2, 0.2% NP-40, and 1 mM PMSF [pH 7.5]), rotated for 10 min at 4 oC, and pelleted by centrifugation at 1,500×g for 5 min. The pellet was resuspended in 1 ml of LB2 (20 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 1 mM PMSF [pH 8.0]) and rotated for 10 min at 4 oC.
The nuclei were pelleted by centrifugation at 1,500×g for 5 min at 4 oC and lysed in 400 ml of LB3 (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1mM PMSF). The lysed nuclei were sonicated using the following conditions: Bioruptor with high power output; 10 cycles (30 s ON and 60 s OFF at 4 oC).
Protein-DNA complexes were immunoprecipitated for 15 hrs at 4 oC using 4 μg of antibodies bound to 50 μl of Dynabeads Protein G (Invitrogen). Immunoprecipitates were sequentially washed with 200 μl of Low salt buffer (0.1 % SDS, 1 % TritonX100, 2 mM EDTA,20 mM Tris, and 150 mM NaCl [pH 8.0]), High salt buffer (0.1 % SDS, 1 % TritonX100, 2 mM EDTA,20 mM Tris, and 500 mM NaCl [pH 8.0]), RIRA buffer (50 mM HEPES-KOH, 0.5 M LiCl, 1 mM EDTA, 0.5% Na-Deoxycholate, and 1% NP-40 [pH 7.4]), and TE buffer (10 mM Tris and 10 mM EDTA [pH 8.0]). For collection of the protein-DNA complexes, beads were resuspended with 50 μl of elution buffer (100 mM NaHCO3, 1 % SDS, and 10 mM DTT). The crosslink for both the immunoprecipitated and input DNA fragments was reversed by incubating at 65 oC for 6 hr. After Proteinase K digestion, the DNA was purified by using PCR purification Kit (Qiagen), and dissolved with TE buffer.
DNA of between 2000 and 350 bp, purified using an Agencourt AMPureXP Kit, was sequenced on a Life Technologies SOLiD 5500xl platform to generate single-end 50 bp reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB 5500xl Genetic Analyzer |
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| Data processing |
ChIP-seq reads were aligned on mm9 genome assembly using bowtie-0.12.8 with "-n 3 -m 1" settings.
2kb binned reads in every 1kb were counted and created BED and WIG files using sql and perl based scripts.
Genome_build: mm9
Supplementary_files_format_and_content: TDF files were created using IGVTools-2.3.5 with "-w 25 -e 250" settings.
Supplementary_files_format_and_content: Bed files were using perl based script.
Supplementary_files_format_and_content: Wig files were using perl based script.
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| Submission date |
Aug 28, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Yukihiro Yabuta |
| E-mail(s) |
yabyab@anat2.med.kyoto-u.ac.jp
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| Organization name |
Kyoto University, Graduate school of medicine
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| Department |
Anatomy and Cell Biology
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| Street address |
Yoshida-Konoe-cho, Sakyo-ku
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| City |
Kyoto |
| State/province |
Kyoto |
| ZIP/Postal code |
606-8501 |
| Country |
Japan |
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| Platform ID |
GPL15907 |
| Series (1) |
| GSE50394 |
A mesodermal factor, T (Brachyury), specifies mouse germ cell fate by directly activating germline determinants |
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| Relations |
| BioSample |
SAMN02338372 |
| SRA |
SRX340832 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1218024_SMAD1_36h_PV.tdf |
303.0 Mb |
(ftp)(http) |
TDF |
| GSM1218024_Smad1_36h_PV_2k_1k.bed.gz |
14.8 Mb |
(ftp)(http) |
BED |
| GSM1218024_Smad1_36h_PV_2k_1k.wig.gz |
2.2 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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