Extended Data Fig. 2: HTGTS V(D)J-seq analysis of V(D)J recombination outcomes in DH–JH+/− line and its mutant derivatives.
From: The fundamental role of chromatin loop extrusion in physiological V(D)J recombination
a, Schematic of the two Igh alleles of the DH–JH+/− v-Abl pro-B line. This C57BL/6, 129/Sv mixed background line was derived by deleting the indicated region from the 129/Sv allele to inactivate it for V(D)J recombination. b, Southern blotting confirmation of allele deletion in the DH–JH+/− line. Done twice with similar results. c, C57BL/6 versus 129/Sv DH usage in parental versus DH–JH+/− lines, as analysed via HTGTS V(D)J-seq (JH1 CE primer). The lack of 129/Sv-specific DHs in DH–JH+/− libraries confirmed the retention of C57BL/6 and absence of 129/Sv allele in this line. d, Bar chart shows utilization frequency of each VH, DH and JH from JH-distal to JH-proximal locales (n = 3 independent libraries). Pie chart shows indicated V(D)J recombination products as percentage of total Igh junctions. Beyond predominant DJH1 junctions, both low-level VHDJH1 joins4,12 and inversional JH(D)JH1 joins38 were detected. There were very low levels of JH1 joins to ‘cryptic RSSs’, or to a different JH-RSS (other) that is likely to occur in extra-chromosomal excision circles13. e, Utilization of each D as a percentage of total DJH1 joins (n = 3 independent libraries). f, Strategy for analysis of D-RSS-DN versus D-RSS-UP utilization. The orientation of D coding sequences relative to the JH1 CE primer is preserved in primary and secondary joins for both D-RSS-DN and D-RSS-UP, allowing calculation of the relative utilization of D-RSS-DN versus D-RSS-UP. g, Utilization frequency of D-RSS-DN versus D-RSS-UP in the DH–JH+/− line. h, Effect of DFL16.1-RSS mutations on utilization of D-RSS-DNs versus D-RSS-UPs. Libraries in d, e, g, h were normalized to 40,000 total junctions. Data represent mean ± s.d. Data for DH–JH+/− line in d–g, h are two sets of three repeats each, with the latter done along with DLF16.1 mutants.
