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| Status |
Public on Feb 11, 2010 |
| Title |
IMR90hTert_SNAP45 |
| Sample type |
SRA |
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| Source name |
IMR90hTert_SNAP45
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| Organism |
Homo sapiens |
| Characteristics |
cell type: IMR90hTert
antibody: SNAP45
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| Treatment protocol |
MR90HTert cells were cross-linked for seven minutes in 1% formaldehyde. Following cell and nuclear lysis as described, extracted chromatin was sonicated to an average size of 200-600 base pairs and used for immunoprecipitations. The sonicated chromatin was mixed with various antibodies and incubated on a rotating wheel overnight at 4˚C. Immunoprecipitated material was recovered by addition of 10 µl of protein A agarose beads (pre-blocked with 10µg/ml of BSA and salmon sperm DNA) and incubation for 1 h at room temperature on a rotating wheel. The beads were then washed with dialysis and wash buffer {O'Geen, 2006 #64}. De-crosslinking, RNase A and proteinase K treatments, and DNA purification were conducted as described {O'Geen, 2006 #64}.
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| Growth protocol |
The chromatin immunoprecipitations (ChIPs) were performed with exponentially growing IMR90HTert cells, which are diploid fibroblast-like cells established from fetal female lung tissue and transfected with the gene coding for human Telomerase reverse Transcriptase.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
At least 10 ng was used to prepare sequencing libraries with the Illumina ChIP-Seq Sample Preparation Kit according to the protocol supplied with the reagents. Aliquots of the sequencing libraries were then loaded into Genome Analyzer flow cells and sequenced with the Genome Analyzer II (Illumina) using the 36 Cycle Sequencing Kit v2 (Catalog number FC-204-2036). As controls, aliquots of the input DNA (collected after the crosslinking and sonication steps) were also subjected to ultra high throughput sequencing.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
MR90HTert stable cell line expressing the human SNAP45 fused to the Tobacco etch virus (TEV), the Flag epitope, and the biotin acceptor domain (BAD) growing DMEM, 10% FBS, and 0,1% of non essential amino acids, were cross-linked for 7 min by addition of 1% of Formaldehyde (Sigma, Cat. No. F-1635). Crosslinking was stopped by addition of glycine to a final concentration of 0.125 M, followed by 5-min incubation at room temperature. Fixed cells were washed twice with cold PBS and harvested in cold PBS containing protease inhibitors. Cells were pelleted by centrifugation, suspended in 1mL of Cell lysis buffer (5mM Pipes at pH 8.0, 0.5% NP40, 85mM KCl, 1mM DTT and protease inhibitors (sigma N° 8340)), and incubated for 10 min on ice. Cells were then pelleted by centrifugation, resuspended in 200 µL of Nuclear lysis buffer (50mM Tris-Cl pH 8.0, 10mM EDTA, 1% SDS, 1mM PMSF, 1mM DTT and protease inhibitors) and incubated for 30min at room temperature. Nuclear extraction was diluted 1:5 with salty buffer (250mM NaCl, 25mM Hepes pH 7.9, 2mM DTT, 0,5 mM PMSF). DNA was disrupted by sonication, yielding genomic DNA fragments of a size of 200–600 bp. Crude chromatin was cleared by centrifugation at 16’000xg for 20 minutes. 500µl of cleared chromatin was next incubated overnight with 30µl of Dynabeads M280-streptavidin (Invitrogen N° 112.05D). Beads were then washed twice with SDS buffer (20mM Hepes pH 7.9, 200mM NaCl, 0.5mM EDTA, 10% glycerol 1mM DTT, 10.5mM PMSF, 2% SDS) followed by two washes with IP wash buffer (100mM Tris at pH 8.0, 500mM LiCl, 1% NP-40, 1% deoxycholic acid), and two washes with TEV-buffer (20mM Hepes pH 7.9, 200mM NaCl, 0.5mM EDTA, 10% glycerol 1mM DTT, 0.5mM PMSF). Samples were then resuspended in 100µl TEV-buffer containing 1unit of TEV enzyme; incubated for 3 hours at room temperature. Beads were concentrate with a magnet and supernatant was collected as first elution. 100µl of TEV-buffer was added to beads and supernatant was pooled with the first elution, then 2µl of DNase-free RNase-A (10mg/ml) was added and samples were decrosslinked by an overnight incubation at 65 °C. Proteinase K and 5X Proteinase K buffer (50mM Tris at pH 7.5, 25mM EDTA, 1.25% SDS) were then added to the samples and incubate at 37°C for 2 hours. DNA from the samples was purified using Gene PCR cleanup columns (Sigma, NA-1020). Purified DNA was recovered in 50µl of elution buffer.
IMR90hTert_SNAP45_run41_lane_2_175-225
IMR90hTert_SNAP45_run41_lane_3_225-275
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| Data processing |
Alignment : The sequenced tags were mapped onto the unmasked genome build hg18 using the fetchGWI software and allowing 1000 repeats on the genome. The tags mapping more than once onto the genome were then attributed a weight corresponding to the number of times the tag was sequenced divided by the number of genomic matches . The files with these weighted repeated tags as well as the unique tags were then used for peak detection
Peak detection : peaks were determined with the sissrs software (www.rajajothi.com/sissrs/). Peaks common to the control input material and the ChIP material, as well as peaks mapping in satellite and micro-satellite repeats and peaks mapping in 18 and 28S rRNA sequences, were eliminated from the analysis. This first analysis resulted in the identification of peaks. The obtained peaks were then quantified using the protocol described in the paper describing this dataset
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| Submission date |
Sep 21, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Nicolo Riggi |
| Organization name |
CHUV
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| Department |
Département de Pathologie Expérimentale
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| Lab |
Institut universitaire de pathologie
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| Street address |
Bugnon 25
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| City |
Lausanne |
| State/province |
VD |
| ZIP/Postal code |
1011 |
| Country |
Switzerland |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE18184 |
Genome-wide localization of the RNA polymerase III transcription machinery in human cells |
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| Relations |
| SRA |
SRX016559 |
| BioSample |
SAMN00008677 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM454599_SNAP45_peaks.bedgraph.gz |
390.4 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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