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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 10, 2013 |
| Title |
Tfe3_ctrl_ChIPSeq |
| Sample type |
SRA |
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| Source name |
ctrl.2, 2i
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| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic stem cell
passage: 14
shRNA: control
chip antibody: Tfe3 (Santa Cruz, sc-5958)
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| Growth protocol |
ES cells were cultured in N2B27 (NDiff N2B27 base medium, Stem Cell Sciences Ltd) supplemented with small-molecule inhibitors PD03 (1uM, PD0325901), CHIR (3uM, CHIR99021) on gelatin coated tissue culture flasks.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ES cells were fixed for 10 min in 1.1% formaldehyde, neutralized with 1M glycine, collected in cold PBS and incubated for 10 min on ice in swelling buffer (50mM HEPES pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40 and 0.25% Tx-100). Nuclei were pelleted, washed and resuspended in shearing buffer (50mM Tris pH 8.0, 10mM EDTA and 1% SDS). Lysates were sonicated using a Bioruptor (Diagenode). Lysates were diluted 1:10 in ChIP dilution buffer (50mM Tris pH 8.0, 167mM NaCl, 1.1% Tx-100 and 0.11% Na-Deoxycholate), pre-cleared for 2h over ProteinG sepharose beads (Amersham) and incubated overnight with 2g Tfe3 (Santa Cruz, sc-5958) or isotype IgG antibodies (Santa Cruz, sc-2025). Lysates were then incubated for 1h with blocked ProteinG sepharose beads, washed 6 times and eluted twice with elution buffer (1% SDS and 0.1M NaHCO3) for 15 min shaking at room temperature. Samples were incubated overnight at 65°C to reverse the cross-linking and purified using QIAquick PCR Cleanup kit (Qiagen). ChiP-Seq libraries were generated using NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (NEB) using 10 amplification cycles.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Data processing |
Basecalls were performed using CASAVA version 1.4
36-nt reads were aligned with Bowtie (vs 0.12.8; to the mouse reference genome (UCSC mm9/NCBI build 37) allowing no mismatches and excluding non-unique mappings. Potential PCR duplicates were removed.
Peaks were called over IgG and Tfe3 shRNA cells with MACS (vs 1.4.2). Processed data files are wig profiles calculated by MACS.
Genome_build: UCSC mm9/NCBI build 37
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| Submission date |
Aug 01, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Joerg Betschinger |
| E-mail(s) |
jb579@cam.ac.uk
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| Organization name |
Wellcome Trust and Medical Research Council Stem Cell Institute
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| Street address |
Tennis Court Road
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| City |
Cambridge |
| ZIP/Postal code |
CB2 1QR |
| Country |
United Kingdom |
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| Platform ID |
GPL9185 |
| Series (1) |
| GSE39815 |
Tfe3-bound regions in naive pluripotent mouse embryonic stem (ES) cells |
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| Relations |
| SRA |
SRX172948 |
| BioSample |
SAMN01096622 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM979714_Tfe3_ctrl_peaks.wig.gz |
3.1 Gb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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