Extended Data Fig. 2. Dependence of V_H-DJ_H recombination on chromatin looping and V_H gene orientation.

a, ChIP-seq analysis of CTCF binding in the distal 890-kb region in short-term cultured pro-B cells from Igh ^{890-inv/890-inv} and Igh ^+/+ mice. Vertical bars indicated CTCF peaks defined by ‘peak calling’. The ChIP-seq data of the Igh ^890-inv allele were aligned on the wild-type Igh sequences. One of two experiments per genotype is shown. b, Igh positions of the 3C-seq viewpoints, which are referred to by the nearest V_H gene, the hypersensitive site 5 (HS5) in the 3’CBE region or the CBE array insertion (Supplementary Table 1c). c, Interactions from the 5’ (V_H1-86; left) and 3’ (HS5; right) viewpoints in short-term cultured pro-B cells from Igh ^{890-inv/890-inv} Rag2 ^–/– and Igh ^+/+ Rag2 ^–/– mice. The 3C-seq reads along the Igh locus (upper two panels) are shown as RPM values with the respective Q values, which were calculated by the r3Cseq program based on two 3C-seq experiments per genotype. The lower panel shows the fold-change of the RPM values for interaction differences of > 2-fold (dashed line) between experimental (Exp) Igh ^{890-inv/890-inv} Rag2 ^–/– and control (Ctrl) Igh ^+/+ Rag2 ^–/– pro-B cells. d, Schematic diagram summarising the loop formation at the Igh ^890-inv allele in pro-B cells. The CBEs with their orientation are indicated by red and blue arrowheads, V_H genes by grey arrows and loops by arches. The new loop domain is indicated in red. e, Schematic diagram of the Igh ^Δ890, Igh ^V8-8 and Igh ^V8-8-inv alleles. The Igh ^V8-8 and Igh ^V8-8-inv alleles were generated by insertion of the V_H8-8 gene with 500 bp of its 5’ and 3’ flanking sequences (lacking any CBE) in the forward or reverse orientation at the deletion point (117,126,667 / 116,237,220; mm9, Chr. 12) of the Igh ^Δ890 allele (see Methods). The distances from the 3’ end of the V_H8-8 gene to the next CBEs and V_H genes are indicated. The sequence of the inserted V_H8-8 with its flanking DNA sequences is shown in Supplementary Table 1b. f, RNA-seq profile of the Igh locus in immature B cells from the bone marrow of Igh ^V8-8/Δ890, Igh ^{V8-8-inv/Δ890} and Igh ^+/Δ890 mice. The V_H8-8 gene position and Igh annotation are indicated. The data of one of two RNA-seq experiments per genotype is shown. g, V_H8-8 mRNA expression in immature B-cells of the indicated genotypes is shown as mean TPM (transcripts per million) value. Each dot corresponds to one experiment.