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| Status |
Public on Jun 28, 2017 |
| Title |
ChIP_seq_TOP2A_MEF |
| Sample type |
SRA |
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| Source name |
Mouse embryonic fibroblasts
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| Organism |
Mus musculus |
| Characteristics |
cell type: Mouse embryonic fibroblasts
genotype: WT
strain: C57BL/6
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde (Sigma) for 10’ at 37°C. Fixation was quenched by addition of glycine (Sigma) at a final concentration of 125 mM. Twenty million fixed cells were washed with PBS and resuspended in 2 ml of RIPA buffer (10 mM Tris [pH 7.6], 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 1× Complete Mini EDTA free proteinase inhibitor (Roche)) or stored at −80°C until further processing. Sonication was performed using the Covaris S2 sonicator at duty cycle 20%, intensity 5, cycle/burst 200 for 30 min or using the Branson sonifier at amplitude 35%, 12 cycles of 20” sonication and 30” of pause. Ten microgram of respective antibody was incubated with 40 μl of Dynabeads Protein A (or G) for 15 min at room temperature. Antibody-bound beads were added to 1 ml of sonicated chromatin, incubated at 4°C overnight, and washed twice with RIPA buffer, twice with RIPA buffer containing 0.3M NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate), once with TE (pH 8.0) plus 0.2% Triton X-100, and once with TE (pH 8.0). Crosslinking was reversed by incubating the beads at 65°C for 4 hr in the presence of 0.3% SDS and 1 mg/ml Proteinase K. ChIP DNA was purified using ChIP DNA Clean & Concentrator kit (Zymo). The library was prepared using the Ovation SP Ultralow library system (Nugen). 75 cycles of sequencing data were acquired on and Nextseq550 (Illumina). Antibodies for ChiP-seq were: anti-RAD21 (ab992, Abcam), anti-CTCF (07-729, Millipore), Topo IIβ Antibody (H-286) (sc13059, SantaCruz) and Topoisomerase II alpha antibody (EP1102Y) (ab52934, Abcam)
libraries were prepared and sequenced following standard Illumina protocols
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 550 |
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| Data processing |
Reads were aligned to the GRCm38p2/mm10 genome assembly.
Alignment done using bowtie version 0.12.7, with the parameters: --best --all --strata -n 2 -m1 -l 50
ChIP-seq peaks were called againts input samples using default parameters.
Genome_build: GRCm38p2/mm10
Supplementary_files_format_and_content: bigwig files were made using Bedtools genomecov function followed by UCSC toolkit function bedGraphToBigWig
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| Submission date |
May 23, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Yaakov Maman |
| Organization name |
Bar-Ilan University
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| Department |
Azrieli Faculty of Medicine
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| Lab |
Genome Instability & Cancer
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| Street address |
Henrietta Szold 8
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| City |
Safed |
| ZIP/Postal code |
1311502 |
| Country |
Israel |
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| Platform ID |
GPL21626 |
| Series (2) |
| GSE99195 |
Genome Organization Drives Chromosome Fragility [ChIP-seq] |
| GSE99197 |
Genome Organization Drives Chromosome Fragility |
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| Relations |
| BioSample |
SAMN07156377 |
| SRA |
SRX2843866 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2635604_ChIP_seq_TOP2A_MEF.bw |
294.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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