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| Status |
Public on Feb 12, 2014 |
| Title |
Homotypic CenH3 (CenH3/CenH3) ChIP-seq |
| Sample type |
SRA |
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| Source name |
Homotypic CenH3 (CenH3/CenH3) ChIP-seq
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| Organism |
Homo sapiens |
| Characteristics |
cell line: eCenH3 HeLa S3
cell type: Cervical cancer
genotype/variation: CenH3 over-expression
chip antibody: CenH3 (Enzo life science, ADI-KAM-CC006-E)
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| Growth protocol |
HeLa S3 overexpressing eCenH3 or the parental cell line (WT) are grown in spinner flask to a concentration of 1.106 cells/ml with a viability of at least 97%.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells are harvested by a low speed centrifugation (500g, 5min, 4°C), washed twice in cold PBS and resuspended in three cell volumes of cold NB buffer (15mM Tris HCl pH 7,5, 15mM NaCl, 60mM KCl, 300mM Sucrose, 1X complete EDTA free (roche diagnostic), 25ng/ml trichostatin A, 10mM beta glycerophosphate, 5mM sodium fluoride, 0,2mM sodium orthovanadate). Four pellet volumes of NB buffer containing 1% NP40 is then added to the cell suspension and incubated 10 min at 4°C on a wheel. After centrifugation at 1200g for 5 min at 4°C nuclei are washed with 3 nuclei volumes of NB buffer and resuspended in ½ nuclei volume of NB buffer. After adding CaCl2 to a final concentration of 2mM nuclei are partially digested with MNase (Nuclease S7 roche diagnostic) to give a range of oligonucleosome. Nuclei are centrifuged 5 min at 16 000g 4°C and the supernatant is dialyzed at least 3h against 1L of lies buffer (Tris HCl pH 7,5 1mM, EDTA pH 8 0,2mM, 1X complete EDTA free (roche diagnostic), 25ng/ml trichostatin A, 10mM beta glycerophosphate, 5mM sodium fluoride, 0,2mM sodium orthovanadate). To extract as much chromatin as possible from the remaining pellet, it is resuspended in one pellet volume of Lysis buffer and dialyzed for at least 3h against 1L of dialysis buffer. After centrifugation 16 000g 4°C 5 min the supernatant of the resuspended pellet is mixed with the first supernatant. The extract is loaded on top of a 5-30% sucrose gradient made in the lies buffer and centrifuged for 16h 29000rpm at 4°C in the SW32 rotor (Beckman Coulter). 1ml fractions are made and 50l is analyzed by agarose gels to determine the nucleosome composition of each fractions. After increasing NaCl concentration to 300mM, Tris/HCl pH 8 to 10mM and NP40 to 0,1%, fractions containing mononucleosomes are pooled and histone are immunoprecipitated with Flag M2 Agarose beads (Sigma Aldrich) for 4h at 4°C or with protein A/G magnetic beads (Thermo scientific) coated with CENP-A (3-19) antibody (Enzo life sciences) overnight at 4°C on a wheel. Beads are washed 3 times with the IP buffer (20mM Tris HCl pH7,5, 300mM NaCl, 0,2mM EDTA pH 8, 0,1% NP40, glycerol 10%, 1X complete EDTA free (roche diagnostic), 25ng/ml trichostatin A, 10mM beta glycerophosphate, 5mM sodium fluoride, 0,2mM sodium orthovanadate) and elute 2 times 1h at 4°C for the flag IP with ½ beads volumes of elution buffer (IP buffer containing 0,4g/ml flag peptide, Sigma Aldrich) 2 times 3h at 4°C for the CENP-A IP with ½ beads volumes of elution buffer (IP buffer containing 0,5g/ml CENP-A 3-19 peptide).
Libraries were prepared according to standard Illumina protocols for paired-end sequencing on the HiSeq2000 machine
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Merged reads were mapped to the RepeatMasked human genome (Hg19) using Bowtie (v0.12.7) allowing up to 3 mismatches and requiring only unique matches (-v 3, -m 1) using the first 50bp of each read.
Peaks were called for ChIP datasets using SICER (v1.0.3) using 200bp sliding windows and merging islands located with 400bp with FDR of 1e-5.
Read density wig files were created using FindPeaks4 (v4.0.13)
Genome_build: hg19
Supplementary_files_format_and_content: Wig files contain read density across the human genome. Enriched peaks are in BED format
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| Submission date |
Jun 07, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Genevieve Almouzni |
| E-mail(s) |
genevieve.almouzni@curie.fr, adam.woolfe@curie.fr
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| Phone |
+33156246701
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| Organization name |
Institut Curie
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| Department |
Nuclear Dynamics and Genome Plasticity
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| Lab |
Chromatin dynamics
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| Street address |
26 Rue D'Ulm
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| City |
Paris |
| State/province |
Ille De France |
| ZIP/Postal code |
75005 |
| Country |
France |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE42951 |
Genome-wide maps of WT and over-expressing CenH3/CENP-A in Human HeLa S3 cells |
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| Relations |
| BioSample |
SAMN02192680 |
| SRA |
SRX297969 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1155628_Homotypic_CenH3_hg19.wig.gz |
189.7 Mb |
(ftp)(http) |
WIG |
| GSM1155628_Homotypic_CenH3_peaks_hg19.bed.gz |
275.6 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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