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| Status |
Public on Aug 20, 2024 |
| Title |
TDP2seq_Ecoli_DNA_SPO11_WT_rep2 |
| Sample type |
SRA |
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| Source name |
Mus musculus, Escherichia coli DNA
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| Organism |
Escherichia coli |
| Characteristics |
tissue: Escherichia coli genomic DNA digested by SPO11 in vitro
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Testis cells from adult mice were obtained as described previously (ref. 1,2). Cells were embedded in plugs of low-melting point agarose to protect DNA from shearing. For in vitro SPO11 treated plasmid or genomic DNA samples, the treated DNA was mixed with wild type B6 (C57BL/6J) mouse testis cells right before plug formation to provide carrier DNA during library preparation. Plugs were treated with SDS, Proteinase K and RNAse as described previously (ref. 1,2). In-plug removal of covalently bound SPO11 using human TDP2 was performed as described with minor modifications (ref. 3). In brief, plugs were equilibrated in TDP2 buffer (0.05 M Tris-HCl pH 8, 0.15 M NaCl, 10 mM Mg2Cl, 0.5 mM Dithriothreitol, 30 μg/ml BSA, 2 mM ATP) and incubated with purified human TDP2 protein (490 pmol per plug, TopoGEN) at 37°C for 30 min according to the TopoGEN protocol. DNA ends were filled-in with T4 DNA polymerase (New England Biolabs) and ligated to biotinylated adaptors as described previously (ref. 1,2,4,5). To minimize the loss of small plasmid fragments through diffusion out of the plugs, the washing step after ligation of the first adaptor was reduced from overnight to 1h for samples containing plasmid DNA. After ligation to biotinylated adaptors, DNA was purified from the agarose, sheared by sonication, purified with streptavidin, ligated to second-end adaptors, amplified, and sequenced as described previously with minor modifications (ref. 1,2,4,5). For end repair after DNA shearing, NEBNext End repair Kit (New England Biolabs) was used. (ref. 1) Yamada et al., Molecular structures and mechanisms of DNA break processing in mouse meiosis. Genes Dev 34, 806-818 (2020). (ref. 2) Kim et al., Optimized methods for mapping DNA double-strand-break ends and resection tracts and application to meiotic recombination in mouse spermatocytes. bioRxiv 2024.08.10.606181; https://doi.org/10.1101/2024.08.10.606181, (2024).(ref. 3) Gittens et al., A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome. Nat Commun 10, 4846 (2019). (ref. 4) Mimitou et al., S. A global view of meiotic double-strand break end resection. Science 355, 40-45 (2017). (ref. 5) Mimitou et al., S1-seq Assay for Mapping Processed DNA Ends. Methods Enzymol 601, 309-330 (2018).
After quality control by Agilent BioAnalyzer, libraries were pooled equimolar and run on an Illumina NextSeq 1000 sequencer. A spike-in of PhiX was added to the run to increase diversity when necessary and for quality control purposes.
Reads were trimmed and filtered by Trim Galore version 0.6.10 with the arguments --paired --length 15 <http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/>.
Sequencing reads were mapped against the pUC19 plasmid sequence, Escherichia coli (ASM584v2), Saccharomyces cerevisiae (sacCer3) or mouse (mm10) reference sequence by bowtie2 version 2.3.5 (ref. 1) with the arguments -N 1 -X 1000. (ref. 1) Langmead et al., Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10, R25 (2009). Uniquely and properly mapped reads (MAPQ ≥ 20) were extracted by samtools version 1.19.2 with the arguments -u -f 2 -q 20 <http://www.htslib.org/>.
Reads were counted at which a nucleotide next to biotinylated adaptor DNA was mapped. Maps were analyzed using R versions 4.2.3 and 4.3.2 <http://www.r-project.org>.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 1000 |
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| Description |
For mapping in vitro SPO11 cleavage sites, Escherichia coli genomic DNA digested by SPO11 in vitro was embedded in plugs together with wild type C57BL/6J mouse testis cells (0.5–1 million cells per plug), whose DNA acted as carrier during library preparation
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| Data processing |
Reads were trimmed and filtered by Trim Galore version 0.6.10 with the arguments --paired --length 15 <http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/>
Reads were mapped against the pUC19 plasmid sequence, Escherichia coli (ASM584v2), Saccharomyces cerevisiae (sacCer3) or mouse (mm10) reference sequence by bowtie2 version 2.3.5 (ref. 1) with the arguments -N 1 -X 1000. (ref. 1) Langmead et al., Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10, R25 (2009). Uniquely and properly mapped reads (MAPQ ≥ 20) were extracted by samtools version 1.19.2 with the arguments -u -f 2 -q 20 <http://www.htslib.org/>.
Reads were counted at which a nucleotide next to biotinylated adaptor DNA was mapped. Maps were analyzed using R versions 4.2.3 and 4.3.2 <http://www.r-project.org>.
Top- and bottom-strand peaks were separately called as nucleotide positions with >3,000, >10 and >15 RPM of strand-specific read counts using averaged pUC19, E. coli, and S. cerevisiae TDP2-seq data, respectively. Positions with less read counts than those located at –1 and +1 positions on top and bottom strands, respectively, were not defined as peaks because those reads could be false-positively enriched due to incomplete fill-in of 5-prime overhangs at DNA ends before adaptor ligation.
Assembly: mm10, pUC19, ASM584v2, SacCer3
Supplementary files format and content: bigWig files contain read counts at positions of DNA double-strand break ends. Reads were not normalized to RPM (reads per million mapped). Letter "F" or "R" in sample names indicates that reads were mapped on forward or reverse strand, respectively.
Supplementary files format and content: bed files contain peak locations. If only one file per experiment is submitted, peaks were determined based on average profiles of the two replicates.
Library strategy: TDP2-seq
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| Submission date |
Aug 20, 2024 |
| Last update date |
Aug 20, 2024 |
| Contact name |
Meret Arter |
| E-mail(s) |
arterm@mskcc.org
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| Organization name |
Memorial Sloan Kettering Cancer Center
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| Street address |
430 E 67th Street
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| City |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL33919 |
| Series (1) |
| GSE275291 |
Reconstitution of SPO11-dependent double-strand break formation |
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| Relations |
| BioSample |
SAMN43274258 |
| SRA |
SRX25775552 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8474253_TDP2seq_Ecoli_SPO11_WT_peaks.rpm.summit.10.bed.gz |
36.9 Kb |
(ftp)(http) |
BED |
| GSM8474253_TDP2seq_Ecoli_SPO11_WT_rep2.F.bw |
314.2 Kb |
(ftp)(http) |
BW |
| GSM8474253_TDP2seq_Ecoli_SPO11_WT_rep2.R.bw |
315.9 Kb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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