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| Status |
Public on Aug 19, 2019 |
| Title |
dCas9Block_DpnII_iEmubait_rep2 |
| Sample type |
SRA |
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| Source name |
v-Abl transformed pro-B cell line
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| Organism |
Mus musculus |
| Characteristics |
genotype: JH-del, dCas9 expressing, Sg1-sgRNA expressing, Emu-Bcl2, RAG2 deficient
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| Treatment protocol |
3 uM STI-571 treated for 4 days
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| Growth protocol |
RPMI1640+15% FBS
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Briefly, 10 million cells were cross-linked with 2% formaldehyde at room temperature for 10 minutes, quenched by glycine (final 0.125M), and then lysed with cold cell lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, one tablet of protease inhibitor in 50 ml) on ice for 10 min. Nuclei were subjected to 0.3% SDS treatment (37oC, 1 hour) and 1.8% Triton X-100 treatment (37 oC, 1 hour) successively, followed by overnight digestion with indicated restriction enzyme (700 units) at 37oC. Restriction enzyme was inactivated at 65oC for 20 min and samples were subjected to ligation under diluted condition with T4 DNA ligase (100 units, NEB, M0202L). Ligated chromatin was de-crosslinked with Proteinase K (56oC, O/N) and treated with RNaseA (37oC, 1 hour). DNA was purified via phenol/chloroform extraction and resuspended in 200 ul 1XT.E. buffer. DNA templates were then subjected to 3C-HTGTS library preparation.
3C-HTGTS libraries were prepared using linear-amplification PCR-mediated HTGTS.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
DpnII digestion, aligned to mm9 genome
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| Data processing |
Library strategy: 3C-HTGTS
Sequencing reads were de-multiplexed and adapter sequence trimmed using the fastq-multx tool from ea-utils (http://code.google.com/p/eautils/) and the SeqPrep utility (https://github.com/jstjohn/SeqPrep) respectively.
Reads were mapped to the mm9 reference genome using Bowtie2 (http://bowtiebio.sourceforge.net/bowtie2/manual.shtml) with the top fifty alignments reported that had an alignment score above 50, representing a perfect 25nt local alignment.
We used a best-path searching algorithm to select the optimal sequence of alignments that describe the read’s composition. Aligned reads were filtered on the following conditions: (1) reads must include both a bait alignment and a prey alignment and (2) the bait alignment cannot extend more than 10 nucleotides beyond the targeted site. For vector controls and offset nicking with multiple sites, the longest targeted site was used.
We compared discarded alignments to the selected prey alignment; if any of the discarded alignments surpassed both a coverage and score threshold with respect to the prey alignment, the read was filtered due to low mapping quality.
To remove possible mispriming events and other artifacts, the bait alignment must extend 10 nucleotides past the primer.
Post-filter stringency was applied to remove background-prone junctions with gaps larger than 30nt, bait sequences shorter than 50nt and sequences with microhomology larger than 5 bp.
Genome_build: mm9
Supplementary_files_format_and_content: bedgraph files
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| Submission date |
Aug 10, 2019 |
| Last update date |
Aug 20, 2019 |
| Contact name |
Frederick W Alt |
| E-mail(s) |
jianqiao.hu@childrens.harvard.edu
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| Organization name |
Boston Children's Hospital
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| Department |
PCMM
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| Lab |
Alt
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| Street address |
1 Blackfan Circle
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL21626 |
| Series (2) |
| GSE130214 |
The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination [3C-HTGTS] |
| GSE130224 |
The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination |
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| Relations |
| BioSample |
SAMN12549318 |
| SRA |
SRX6693290 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4026042_dCas9Block_DpnII_iEmubait_rep2.bedgraph.gz |
227.1 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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