Extended Data Fig. 1. Generation and characterization of the Igh ^890-inv allele.

a, Orientation of the CTCF-binding sites in the Igh locus. The CTCF-binding pattern was determined by ChIP-seq of Rag2 ^–/– pro-B cells. The locations of forward (red) and reverse (blue) CTCF motifs detected at the summit of the CTCF peaks are shown together with all predicted CTCF motifs, identified based on the forward and reverse consensus CTCF-binding motifs shown. The annotation of the C57BL/6 Igh locus indicates the distinct V_H gene families (different colours) in the distal, middle and proximal V_H gene regions (Johnston et al. 2006. J. Immunol. 176, 4221-4234) and the 3’ proximal Igh domain containing the D_H, J_H and C_H elements as well as the Eμ and 3’RR enhancers (red). b, Schematic diagram of the Igh ^ifl-890-fl and Igh ^890-inv alleles. The indicated selection cassettes were used for introducing the upstream inverted lox71 site (ifl) and downstream loxP site (fl) by sequential ES cell targeting. c, Flow cytometric analysis of bone marrow cells from Igh ^{890-inv/890-inv} and Igh ^+/+ mice. The relative frequencies of the indicated cell types (defined in Methods) are shown as mean values with SEM. d, Flow cytometric determination of the ratio of immature IgM^b (B6) to IgM^a (129) B cells from Igh ^{890-inv(B6)/+(129)} and Igh ^+(B6)/+(129) mice, which were generated by crossing Igh ^890-inv/+ mice on the C57BL/6 (B6) background with Igh ^+/+ mice of the 129/Sv (129) strain. The rearranged Igh alleles of the C57BL/6 and 129/Sv strains give rise to expression of the IgM^b and IgM^a isotypes, respectively. e, V_H gene expression from the Igh ^890-inv(B6) or Igh ^+(B6) allele in immature IgM^b (B6) B cells sorted from Igh ^{890-inv(B6)/+(129)} or Igh ^+(B6)/+(129) mice, respectively. The RNA-seq profiles are shown with the Igh annotation (see a) and inverted region (bar). One of two experiments is shown. RPM, reads per million mapped sequence reads. f,g VDJ-seq analysis of pro-B cells from the bone marrow of Igh ^{890-inv/890-inv} and Igh ^+/+ mice. f, The percentages of uniquely identified DJ_H and VDJ_H sequences are indicated. g, The relative usage of the distal V_H genes at the Igh 5’ end is shown as mean percentage of all VDJ_H recombination events with SEM. A horizontal bar indicates the 5’ end of the inverted region. Statistical data are shown as mean value with SEM and were analysed either by multiple t-tests (unpaired and two-tailed with Holm-Sidak correction; c,f) or by the Student’s t-test (unpaired and two-tailed; d). n, number of mice (c,d) or experiments (f,g). Each dot corresponds to one mouse.