title: GSM4178015: HTGTS_PB_VK1_117_WT_REP5; Mus musculus; OTHER
library_construction_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).
identifiers.namespace: GEO
identifiers.id: GSM4178015