Fig. 1: RAG-generated extrachromosomal circular DNAs persist throughout mouse B cell development.

a, Schematic of the mouse immunoglobulin kappa (Igk) locus, highlighting the gene segments studied: Vκ16-104, Vκ3-1 and Vκ11-125, which undergo deletional recombination to generate an ESC. All Igl recombination reactions are deletional. Grey and blue triangles represent 12- and 23-RSSs, respectively; blue and purple rectangles represent V and J gene segments, respectively; the orange rectangle represents the constant region exon; the RS element is depicted by a green rectangle. b, Ratio of SJs to recombination junctions (Rec) at sequential stages of B cell development as determined by absolute qPCR for Jκ5-Vκ3-1, Jκ5-Vκ16-104 and Jλ1-Vλ1 and compared with the ratio in pre-B cells, which was set at 1:1 for ease of comparison. n = 3 samples. BM, bone marrow. c, Schematic of the isolation of ESCs from mouse mature B cells. Splenocytes were isolated from six-week-old mice and purified by flow cytometry to obtain IgM⁺ and IgG⁺ B cells (Supplementary Fig. 1); genomic DNA (gDNA) was extracted and digested with RecBCD to remove linear DNA. d, Jκ5-Vκ11-125 and Jκ5-Vκ16-104 ESCs persist as circles in mouse mature B cells (IgM⁺, upper and IgG⁺, lower). The amount of undigested DNA was determined by qPCR. Gapdh (encoded by linear, genomic DNA) was used as a negative control; untreated DNA was a further control (Ctrl). P values were determined by an unpaired, two-tailed Student’s t-test. Comparison versus Gapdh, 95% confidence interval: IgM⁺ Jκ5-Vκ11-125, −71.36 to −13.20; IgM⁺ Jκ5-Vκ16-104, −79.49 to −25.81; IgG⁺ Jκ5-Vκ11-125, −80.44 to −10.43; IgG⁺ Jκ5-Vκ16-104, −88.83 to −37.15. n = 3 samples. e, Rag1 expression levels at different stages of mouse B cell development as determined by quantitative PCR with reverse transcription (RT–qPCR). Data are normalized to Hprt expression levels. NIH3T3 cDNA is a negative control. n = 3 samples. Mean values are shown; error bars represent s.d.

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