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Sample GSM4178036 Query DataSets for GSM4178036
Status Public on Jan 01, 2020
Title HTGTS_Spl_B_VK1_117_REP3
Sample type SRA
Source name Primary splenic B cell
Organism Mus musculus
Characteristics cell type: Primary splenic B cell
genotype: WT
strain: C57BL/6
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.
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).
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).
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina MiSeq
Description HTGTS_Spl_B_VK1_117.tlx
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)
Submission date Nov 19, 2019
Last update date Jan 01, 2020
Contact name Yaakov Maman
Organization name Bar-Ilan University
Department Azrieli Faculty of Medicine
Lab Genome Instability & Cancer
Street address Henrietta Szold 8
City Safed
ZIP/Postal code 1311502
Country Israel
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
BioSample SAMN13329354
SRA SRX7184539

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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