Extended Data Fig. 3: Characterization of Igh alleles with inserted CBE arrays.
From: Wapl repression by Pax5 promotes V gene recombination by Igh loop extrusion
a, Insertion of an array of 20 functional CBEs in forward (red) or inverted (inv, blue) orientation at position 116,242,836 (mm9, Chr. 12) in the IghCBE or IghCBE-inv allele, respectively. The positions of the Igh CBEs used for the generation of the CBE arrays (Supplementary Table 1c) are indicated below a map of all CBEs. b, CTCF binding at the Igh locus, as determined by ChIP-seq analysis of short-term-cultured pro-B cells from the bone marrow of IghCBE-inv/CBE-inv and IghCBE/CBE mice. One of two experiments is shown. c, Flow cytometric determination of the ratio of immature IgMb (B6) to IgMa (129) B cells in the bone marrow of IghCBE-inv(B6)/+(129), IghCBE(B6)/+(129) and Igh+(B6)/+(129) mice (Extended Data Fig. 1d). The IgMb/IgMa ratios are shown relative to that of immature B cells of Igh+(B6)/+(129) mice (set to 1). Statistical data are shown as mean value with s.e.m. and were analysed by one-way ANOVA with Tukey’s post hoc test. d, VH gene expression from the IghCBE-inv(B6), IghCBE(B6) and Igh+(B6) alleles in immature IgMb (B6) B cells sorted from IghCBE-inv(B6)/+(129), IghCBE(B6)/+(129) and Igh+(B6)/+(129) mice, respectively (Extended Data Fig. 1d). The expression (TPM) value of each VH gene is shown as shared (grey) and unique expression of the IghCBE-inv(B6) (blue), IghCBE(B6) (green) and Igh+(B6) (black) alleles. Mean TPM values with s.e.m. were determined by the DESeq2 program and are based on two (IghCBE-inv(B6), IghCBE(B6)) and three (Igh+(B6)) RNA-seq experiments. The VH genes are aligned according to their Igh position. e, f, VDJ-seq analysis of ex vivo-sorted pro-B cells from the bone marrow of Igh+/+, IghCBE/CBE and IghCBE-inv/CBE-inv mice. e, The percentages of uniquely identified DJH and VDJH sequences are shown as mean percentage with s.e.m. f, Pairwise comparisons of VDJ-seq results obtained with Igh+/+ (black), IghCBE/CBE (green) and IghCBE-inv/CBE-inv (blue) pro-B cells. The relative usage of each VH gene was determined as percentage of all VDJH recombination events and is shown as mean percentage with s.e.m. n, number of mice. Each dot corresponds to one mouse (c, e).
