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| Status |
Public on Jul 15, 2016 |
| Title |
Input_Mm |
| Sample type |
SRA |
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| Source name |
Mouse Crypt
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| Organism |
Mus musculus |
| Characteristics |
tissue: intestinal organoid
chip antibody: none
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| Growth protocol |
Mouse intestines were flushed with PBS before being cut open longitudinally. The villi were removed by scraping with a microscope slide. Pieces of approximately 1 cm in length were incubated in 5 mM EDTA/PBS without Ca2+/Mg2+ for 15 min at 4°C per fraction. After incubation, the epithelium was separated by vigorous shaking, and the remaining intestinal tissue was placed in a new tube for collection of the following fraction. Remaining crypts were pelleted and evaluated for purity under the microscope. The two purest fractions were pooled and used as input material for ChIP-seq. All procedures were performed in compliance with local animal welfare laws, guidelines, and policies.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
human cells or mouse intestinal crypts were crosslinked with 1% formaldehyde for 20 min at room temperature. For β-catenin ChIP-seq, cells were crosslinked for 40 min using ethylene glycol-bis(succinimidylsuccinate) (Thermo Scientific) at 12.5 mM final concentration, with the addition of formaldehyde (1% final concentration) after 20 min of incubation. The reaction was quenched with glycine, and the cells were successively washed with PBS, buffer B (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 20 mM HEPES, pH 7.6), and buffer C (0.15 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES, pH 7.6). The cells were then resuspended in shearing buffer (0.3% SDS, 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES, pH 7.6) and sheared using Covaris S2 (Covaris) for 8 min with the following settings: duty cycle, max; intensity, max; cycles per burst, max; mode, power tracking.
Immunoprecipitated chromatin was sheared, end-repaired, sequencing adaptors were ligated and the library was amplified by ligation mediated PCR (LMPCR). After LMPCR, the library was purified and checked for the proper size range and for the absence of adaptor dimers on a 2% agarose gel, barcoded and sequenced on SOLiD/AB sequencer in multiplexed way to produce 50 base pair long reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB SOLiD System 3.0 |
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| Data processing |
Sequencing reads were mapped against the reference genome (mm9) using BWA package
Sequencing reads were mapped against the reference genome (hg19) using BWA package
Multiple reads mapping to same location and strand have been collapsed to single read and only uniquely placed reads were used for peak calling.
Genome_build: mm9, hg19
Supplementary_files_format_and_content: [.wig] file reports peaks
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| Submission date |
Jul 15, 2016 |
| Last update date |
Jul 15, 2016 |
| Contact name |
Jurian Schuijers |
| Organization name |
Hubrecht Insitute
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| Street address |
Uppsalalaan 8
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| City |
Utrecht |
| State/province |
Utrecht |
| ZIP/Postal code |
3584CT |
| Country |
Netherlands |
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| Platform ID |
GPL9318 |
| Series (2) |
| GSE84456 |
Ascl2 acts as an R-spondin/Wnt responsive switch to control stemness in intestinal crypts [ChIP-seq] |
| GSE84457 |
Ascl2 acts as an R-spondin/Wnt responsive switch to control stemness in intestinal crypts |
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| Relations |
| BioSample |
SAMN05402501 |
| Supplementary data files not provided |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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