Figure 1.

The VDJ-Seq Technique

(A) Genomic DNA from sorted pro-B cells (1) containing unknown VDJ_H and DJ_H joins (only VDJ_H depicted) is sonicated to 500 bp (2), end-repaired, A-tailed, and a custom adaptor ligated (3). Primer-extension is performed with forward and reverse primers that hybridize upstream of each J_H gene (4). Following depletion of unrecombined primer-extended DNA with streptavidin beads (4), a second primer-extension is performed extending upstream into VDJ_H or DJ_H recombined sequences from biotinylated primers that hybridize downstream of each J_H (5). After capture with streptavidin beads, two rounds of PCR generate the sequencing library; the first using adaptor-specific paired-end 1 (PE1) and J-specific paired-end 2 (PE2) primers (6), the second using flow-cell PE1 and PE2 primers (7) to generate the library (8).

(B) Differentiation of early B cell progenitors in bone marrow showing when D_H-to-J_H and V_H-to-DJ_H joining occur. Rag1^−/− mice are incapable of V(D)J recombination.

See also Figures S1 and S3.