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| Status |
Public on Apr 24, 2019 |
| Title |
END_seq_MEF_NT (re-used) |
| Sample type |
SRA |
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| Source name |
Activated primary B cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: WT
cell type: Abelson-transformed pre-B cell
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| Treatment protocol |
Pre-cells were arrested in G1 by treatment with 3 mM imatinib for 48 hr and then treated with etoposide for the times indicated. MEFs, after 10 days in confluence and serum starvation (2% charcoaltreated FBS) were treated with 50uM of etoposide during 30 minutes and 2 hours and 30 minutes. HCT116 cells were arrested in G1 by the treatment with 20uM of lovastatin for 24 hours and treated with auxin for 4 hours before 50uM etoposide treatment for 30 minutes. For transcription inhibition or stimulation, and proteasome inhibition drug pre-treatment was performed for 2 hours with 150 mM DRB (D-ribofuranosylbenzimidazole, Sigma) or 2uM of epoxomicin (Sigma) prior to etoposide treatment. Drugs were maintained during the treatment with etoposide.
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| Growth protocol |
Abelson pre-B cell lines were cultured DMEM supplemented 10% FBS, with 2 mM L-glutamine, 1 mM Sodium piruvate, non-essential amino acids, 0.05 mM β-mercaptoethanol, penicillin (100 U/ml) and streptomycin (100 U/ml) (Invitrogen). MEFS were cultured and floxed alleles deleted as described (Busslinger et al., 2017). Mature WT and Top2b-/- B-cells were generated by bone marrow reconstitution of lethally irradiated recipient mice with fetal liver cells derived from WT and Top2b-/- embryos, respectively. Recipient mice were irradiated with 950 rads, 4 hours before injection of 3 million donor fetal liver cells. Between 6 to 12 weeks after reconstitution mature resting B cells were isolated from recipient mouse spleen with anti-CD43 MicroBeads (Miltenyi Biotech). B cells were activated with LPS (25 mg/ml; Sigma), IL-4 (5 ng/ml; Sigma) and RP105 (0.5 mg/ml; BD) for 12 hr as described (Callen et al., 2013) and treated for 30 minutes with 50uM of etoposide.HCT116 cells were obtained from (Natsume et al., 2016), arrested in G1 by the treatment with 20uM of lovastatin for 24 hours and treated with auxin for 4 hours before 50uM etoposide treatment for 30 minutes.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Single cell suspensions of pre-B cells (40 million per agarose plug), MEFS (4 million per agarose plug), and HCT116 (15 million per 2 agarose plugs) were washed in PBS, embedded in agarose and transferred into plug molds (Bio-Rad CHEF Mammalian Genomic DNA plug kit) and processed as previously described (Canela et al., 2016)
END-seq was performed as described in Canela et al., 2017. Agarose plugs were made using the Bio-Rad CHEF Mammalian Genomic DNA plug kit. Embedded cells were lysed and digested using Proteinase K (50º C, 1 hour then 37ºC for 7 hours). Plugs were rinsed in TE buffer and treated with RNase A at 37ºC, 1 hour. The next enzymatic reactions were performed with the DNA in agarose plugs to prevent shearing. DNA ends were blunted for 1 hour at 37C with Exonuclease VII followed by Exonuclease T (NEB) for 1 hour at 25ºC to detect all TOP2 lesions, or only with Exonuclease T (NEB) 1 hour at 25C to detect only DSBs, "ExoT END-seq". After blunting, A-tailing was performed, followed by ligation of “END-seq hairpin adaptor 1”, 5′-Phos-GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGUU[Biotin-dT]U[Biotin-dT]UUACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3′ [*phosphorothioate bond], using NEB Quick Ligase. Agarose plugs were then melted and dialyzed and DNA was sonicated to a median shear length of 170bp using Covaris S220 sonicator. DNA was ethanol-precipitated and dissolved in 70 ul TE buffer. Biotinylated DNA was isolated using MyOne Streptavidin C1 Beads (ThermoFisher #650-01), followed by end repair and dA-tailing. The second end was ligated to “END-seq hairpin adaptor 2”, 5′-Phos-GATCGGAAGAGCACACGTCUUUUUUUUAGACGTGTGCTCTTCCGATC*T-3′ [*phosphorothioate bond], using NEB Quick Ligase. Hairpins were digested using USER (NEB), and the resulting DNA fragments were PCR amplified using TruSeq barcoded primer p5, AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC*T and TruSeq barcoded primer p7, CAAGCAGAAGACGGCATACGAGANNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T, (NNNNNNNN represents barcode and * a phosphothiorate bond). PCR fragments were isolated by size selection from agarose gel, selecting 200-500 bp fragments followed by DNA purification using QIAquick Gel Extraction Kit. Libraries were quantified using KAPA Library Quantification Kit and sequenced using Illumina NextSeq 500 or 550.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
This is duplicated sample record of GSM2635571 for the convenient retrieval of the complete raw data from SRA
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| Data processing |
mouse and human END-seq reads were aligned to the GRCm38p2/mm10 and GRCh37/hg19 genome assemblies,respectively.
Alignment done using bowtie version 0.12.7, with the parameters: --best --all --strata -n 3 -k1 -l 50
END-seq peaks were called using MACS 1.4.3 with the parameters: --nolambda,-- nomodel and –keep-dup=all (keep all redundant reads). For Etoposide-treated END-seq data peak calling, we used the corresponding non-treated samples as control, keeping >10 fold-enriched peaks.
END-seq data from pre-B cells, where cells with ZFN break were spiked-in to the library at a 1:50 (2%) ratio, the RPKM value for each peak was divided by the signal around the spiked-in breaks and then multiplied by 2, to get values as cell-percentage. From this value, X, the #cells per lesion were calculated as 100/X.
Genome_build: GRCm38p2/mm10 for mouse and GRCh37/hg19 for human
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 |
Apr 09, 2019 |
| Last update date |
Apr 24, 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) |
| GSE129524 |
Topoisomerase II-induced Chromosome Breakage and Translocation Is Determined by Chromosome Architecture and Transcriptional Activity [END-seq] |
| GSE129529 |
Topoisomerase II-induced Chromosome Breakage and Translocation Is Determined by Chromosome Architecture and Transcriptional Activity |
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| Relations |
| BioSample |
SAMN11372048 |
| SRA |
SRX5657102 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not provided for this record |
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