Anti igm fitc

For RAG on-target and off-target analysis, single cell suspensions were derived from bone marrows of 4–6-week-old C57BL/6 WT and IgH V_H inversion mice and incubated in Red Blood Cell Lysing Buffer (Sigma-Aldrich, #R7757) to deplete the erythrocytes. B220⁺CD43^hi+IgM^- pro-B cells were isolated by staining with anti-B220-APC (eBioscience, #17–0452-83), anti-CD43-PE (BD PharMingen, #553271), and anti-IgM-FITC (eBioscience, #11–5790-81) antibodies and purified by fluorescence-activated cell sorting (FACS), the purified primary pro-B cells were subjected to HTGTS-V(D)J-seq as described^{13 (link),14 (link)}.
For 3C-HTGTS, ChIP-seq and GRO-seq, B220-positive WT and IgH V_H inversion primary pro-B cells were separately purified via anti-B220 MicroBeads (Miltenyi, #130–049-501) from individual 4–6-week-old RAG1-deficient mice (WT and IgH V_H locus inversion) and were cultured in opti-MEM medium containing 10% (v/v) FBS plus IL-7 and SCF for 4–5 days as previously described^{35 (link)}. Purified primary pro-B cells from different mice were separately cultured in different Petri dishes. Each sample was double-checked and confirmed by PCR prior to various assays as described below. PCR primers are listed in Supplementary Table 6.

Mononuclear BM cells, B cells after culture or splenocytes were treated 10 minutes with FC block solution (BD Bioscience) then labelled 20 minutes on ice with combination of different anti-mouse antibodies (Abs) as indicated in legends of figures. Abs included anti-B220-eFluor660 (Ebioscience, Clone RA3-6B2), anti-IgM-Fitc (Ebioscience, Clone eB121-15F9), anti-IgG1-PE (Ebioscience, Clone m1-14D12), biotin anti-IgG2a^b (BD Bioscience, Clone 5.7), biotin anti-mouse IgG3 (BD Bioscience, Clone R40-82), anti-CD138-BrillantViolet421 (BD Bioscience, Clone 281-2), anti-CD43-PE (BD biosciences, Clone S7), anti-CD45.1-PeCy7 (BD biosciences, Clone A20) and anti-CD45.2-APC-Cy7 (BD biosciences, Clone 104). Biotinylated primary antibodies were detected by incubation of labeled cells with streptavidin-PE (BD Bioscience). For apoptosis analysis, B cells were first surface labeled for ASC detection, then washed and re-suspended in 1x Binding buffer (Ebioscience) and labeled with anti-AnnexinV-Fitc (Ebioscience). Ten minutes before flow cytometry acquisition, propidium iodide (Ebioscience) was added to all samples for detection and/or exclusion of dead cells. Cells were analyzed with a FACSFortessa or a FACSCanto (BD Bioscience) and Diva (BD Bioscience) or FLowJo (TreeStar) softwares.

Single-cell suspensions from the spleens and livers of CLT and CLT; AID^−/− RDBC chimeras and control mice were stained with each of the following sets of anti-mouse monoclonal antibodies: anti-B220-PE-Cy5 (eBioscience Inc.) and anti-IgM-FITC (eBioscience Inc.); anti-B220-APC (eBioscience Inc.) and anti-Igκ-PE (BD Biosciences); anti-CD19-FITC (BD Biosciences) and anti-CD95/Fas-PE-Cy7 (BD Biosciences); or anti-CD8a-APC (BD Biosciences) and anti-CD4-FITC (eBioscience Inc.). FACS data were acquired using the FACSCalibur Flow Cytometer equipped with CellQuest software (BD Biosciences) and analyzed with FlowJo software (TreeStar).