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| Status |
Public on Jan 01, 2020 |
| Title |
END_seq_PB_WT_REP5 |
| Sample type |
SRA |
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| Source name |
Abelson-transformed pre-B cell
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| Organism |
Mus musculus |
| Characteristics |
cell type: Abelson-transformed pre-B cell
genotype: WT
strain: C57BL/6
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| Treatment protocol |
Pre-cells were arrested in G1 by treatment with 3 mM imatinib for 48 hr.
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| Growth protocol |
Abelson pre-B cell lines and primary mouse bone marrow pre-B cells 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). Primary mouse bone marrow pre-B cells were cultured in the presence of recombinant IL-7 (ThermoFisher).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Single cell suspensions of pre-B cells (30 million per agarose plug), primary mouse bone marrow pre-B cells (10 million per agarose plug), and thymocytes (70 million per 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). Primary mouse bone marrow pre-B cells for END-Seq analyses were obtained from 6-12 week old Vavp-Bcl2-transgenic mice (Ortega-Molina et al., 2015) and cultured in the presence of recombinant IL-7 (ThermoFisher) for 7-10 days at 2 x 106 cells/ml in vitro. To induce cell cycle arrest, cells were resuspended in media without IL-7 and maintained at 2 × 106 cells/ml for 48 hours and harvested for END-seq.
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 25ºC with Exonuclease T (NEB). 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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| Data processing |
Library strategy: END-seq
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 |
Nov 18, 2019 |
| Last update date |
Jan 02, 2020 |
| 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) |
| GSE140635 |
Intra-Vk Cluster Recombination Shapes the Ig Kappa Locus Repertoire [END-seq] |
| GSE140677 |
Intra-Vk Cluster Recombination Shapes the Ig Kappa Locus Repertoire |
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| Relations |
| BioSample |
SAMN13326829 |
| SRA |
SRX7182763 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4176060_END_seq_PB_WT_REP5.bw |
86.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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