HTGTS for Linear RAG Scanning Mediates Editing of Igκ Variable Region Repertoires
HTGTS for Linear RAG Scanning Mediates Editing of Igκ Variable Region Repertoires
Pre-B cells were isolated from 129SV WT or Cer/Sis-deleted mouse bone marrow by FACS sorting for B220+ CD43low IgM- cells Immature B cells were isolated from iPSC chimera-derived bone marrow by FACS sorting for B220+ CD19+ IgM- IgG1+ cells v-Abl cell lines were cultured in RPMI1640 media with 15% FBS RAG complementation was performed by infecting v-Abl cell lines with RAG2 retrovirus followed by selection with puromycin G1-arrest was performed by treating v-Abl cell lines with 3uM STI for 4 days DNA was lysed overnight at 56degC in a lysis buffer containing: 200mM NaCl, 0.2% SDS, 10mM Tris-HCl pH7.4, 2mM EDTA pH8.0, 200ug/ml proteinase K, precipitated by isopropanol and dissolved in buffer containing 10mM Tris-HCl pH7.4, 0.5mM EDTA pH8.0 HTGTS-V(D)J-seq libraries were prepared using linear-amplification PCR-mediated high-throughput genome-wide translocation sequencing protocol with indicated bait primers