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| Status |
Public on Jan 01, 2020 |
| Title |
HTGTS_Spl_B_VK1_117_REP3 |
| Sample type |
SRA |
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| Source name |
Primary splenic B cell
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| Organism |
Mus musculus |
| Characteristics |
cell type: Primary splenic B cell
genotype: WT
strain: C57BL/6
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| Treatment protocol |
Abelson pre-B cells were arrested in G1 by treatment with 3 mM imatinib for 48 hours and harvested for HTGTS. For detecting translocation from Cas9/gRNA DSBs, gRNA was design to generate DSBs 654 bp downstream of the Vk1-117 bRSS breakage site. HTGTS primer was design that allowed 5' broken ends of these DSBs to be used as a bait. WT abelson pre-B cells were treated with 3 μM imatinib at a concentration of 5x105 cells/ml for 30 hours followed by nucleofection of pX330-Cas9-Vk1-117-CRSPR plasmid (15μg) into 20 million G1-arrested cells (2 reactions; 10 million cells each reaction) using X-001 program of Amaxa Nucleofector II (Lonza) with Human B cell Nucleofector kit (Lonza). Transfected cells were cultured in DMEM medium with 15% (v/v) FBS plus 3 μM imatinib for 3 more days before harvested for genomic DNA.
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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).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
10 million of Abelson pre-B cells, primary mouse bone marrow pre-B cells, splenic IgM positive B cells, and human PBMC B cells were used for HTGTS analyses. Primary mouse bone marrow pre-B cells were obtained from 6-12 week old wild-type, C57BL/6 mice with flow cytometry cell sorting on a FACSAria II cell sorter (BD Biosciences) using a combination of anti-B220, anti-CD43, anti-CD24 and anti-IgM antibodies (ThermoFisher/eBioscience) (Lin et al., 2016). IgM positive mature resting B cells were isolated from C57BL/6 mice spleen with anti-CD43 MicroBeads (Miltenyi Biotech). Human peripheral blood B cells were isolated from deidentified, donated PBMC using human B cell isolation kit (STEMCELL Technologies 17954).
HTGTS was performed as described (Hu et al., 2015). Genomic DNA was extracted from splenic B cells, isolated B cells from human PBMC, or v-Abl cells arrested in G1 for 2 days by treatment with 3 mM of STI-571. Briefly, 20-50 ug of DNA was fragmented via sonication on a Diagenode bioruptor and subjected to linear PCR amplification with a biotinylated primer. Single-stranded PCR products were purified via Dynabeads MyONE C1 streptavidin beads (Life Technologies, 65002) and ligated to bridge adaptors. Adaptor-ligated products were amplified via nested PCR with indexed locus-specific primers and primer annealed to adaptor. The PCR products were further tagged with Illumina sequencing adaptor sequences, size-selected (fragment size 500 – 1,000 bp) via gel extraction and loaded to Illumina MiSeq for paired-end 250 bp sequencing. For translocation analysis, the standard LAM-HTGTS bioinformatic pipeline was used (Hu et al., 2015).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Description |
HTGTS_Spl_B_VK1_117.tlx
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| Data processing |
Library strategy: HTGTS
Reads were aligned to the GRCm38p2/mm10 and GRCh37/hg19 genome assembly
Sites that underwent chromosomal translocations to the bait sites were identified as described (Hu et al., 2016).
Genome_build: GRCm38p2/mm10, GRCh37/hg19
Supplementary_files_format_and_content: bigwig files were made using Bedtools genomecov function followed by UCSC toolkit function bedGraphToBigWig . Supplementary_files_format_and_content: tlx files were generated using HTGTS pipeline (Hu et al. 2016)
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| Submission date |
Nov 19, 2019 |
| Last update date |
Jan 01, 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 |
GPL16417 |
| Series (2) |
| GSE140672 |
Intra-Vk Cluster Recombination Shapes the Ig Kappa Locus Repertoire [HTGTS] |
| GSE140677 |
Intra-Vk Cluster Recombination Shapes the Ig Kappa Locus Repertoire |
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| Relations |
| BioSample |
SAMN13329354 |
| SRA |
SRX7184539 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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