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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 30, 2015 |
| Title |
ESC+SB_HEB_ChIPseq |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
strain: CGR8
cell type: Embryonic stem cells
passages: 18-25
chip antibody: HEB (Santa Cruz, sc-357, lot G0209)
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| Treatment protocol |
not applicable
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| Growth protocol |
Mouse embryonic stem cells (ESCs) were cultured on gelatin coated plates in the absence of feeder cells. ESC medium was prepared by supplementing knockout DMEM (Invitrogen) with 15% FBS, 1 mM glutamax, 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol and 1000 units of LIF (Millipore). To inhibit TGF-beta signaling pathway, ESCs were treated with 10 µM SB431542 (Tocris Bioscience) for 24 hours. For endoderm differentiation, ESCs were dissociated by TrypLE Express and cultured in serum-free defined differentiation media consisting of 75% Iscove’s modified Dulbecco’s medium and 25% Ham’s F12 media supplemented with 0.5x of both N2 and B27 (without retinoic acid), 0.05% BSA and 50 µg/ml ascorbic acid (Sigma). At day 2, human Activin A (100 ng/ml; R&D systems) was added to induce endoderm differentiation without dissociation/reaggregation. After 48 hours (day 4), ESC-derived endoderm samples were performed for ChIP-Seq. To inhibit TGF-beta signaling pathway, Activin A was treated with 10 µM SB431542 at day 2, then cultured for 48 hours to get day 4, ESC-derived differentiated samples.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and ChIP-DNA complexes were isolated with antibody.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Catalog # FC-102-1001). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3' to 5' exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of 200~400 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer II following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
ChIP DNA
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| Data processing |
Basecalls performed using CASAVA version 1.8.2
Sequence reads were aligned to the mouse reference genome (mm9; July 2007) using the DNAnexus pipeline.
Peaks called using QuEST 2.4 (Valouev et al., 2008) in the DNAnexus platform (https://dnanexus.com) using a “transcription factor” setting (bandwidth of 30 bp, regions size of 300 bp) and 25-fold ChIP to input enrichment for seeding the regions at less than 0.001 FDR.
Genome_build: mm9
Supplementary_files_format_and_content: peaks called from ChIP samples, in TSV format
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| Submission date |
Dec 11, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Se-Jin Yoon |
| E-mail(s) |
sjyoon@stanford.edu
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| Phone |
650-736-0278
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| Organization name |
Stanford University
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| Department |
Genetics
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| Street address |
300 Pasteur Drive, Alway M311
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL11002 |
| Series (2) |
| GSE53233 |
HEB associates with PRC2 and SMAD2/3 to regulate developmental fates [ChIP-Seq] |
| GSE60286 |
HEB associates with PRC2 and SMAD2/3 to regulate developmental fates |
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| Relations |
| BioSample |
SAMN02441894 |
| SRA |
SRX390394 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1288313_ESC_SB_HEB.txt.gz |
755.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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