Extended Data Figure 2. Genome-wide translocation junctions lack orientation bias. | Statistical analyses for experimental replicates in Fig. 1 and 2. | Orientation-biased joining between I-SceI break in place of Sα and AID-initiated Sμ breaks in CH12F3 cells.

(a, b) Circos plots for translocation junctions across the whole genome from 3’BE (a, n=4) or 5’BE (b, n=3) HTGTS with anti-CD40/IL4 stimulated ΔSμ^2xI/ΔSγ1^2xI B cells.

(c, d) Bar graphs depicting percentage of genome-wide junctions from pooled 3’BE and 5’BE libraries plotted separately mapping in (-) or (+) orientation ± s.d., respectively.

(e) Joining from ΔSγ1^2xI 3’-BE to AID off-target DSBs in Il4ra gene on chr 7.

(f) Bar graph showing the percentage of junctions (average ± s.d.) recovered from IgH^I-96k AID^-/- 3’-BE HTGTS libraries (n=3) at breaksite and the upstream Sμ^1xI prey break as the percentage over the total number of junctions mapped to the 200 kb IgH constant region. Right panel shows the percentage of junctions mapping at Sμ^1xI (average ± s.d.) over the total IgH junctions that are mapped in the deletion (Del) or inversional (Inv) orientation. The numbers above the bar graph (average ± s.d.) denote ratio of deletional versus inversional junctions.

(g) Bar graph showing the percentage of junctions (average ± s.d.) recovered from the IgH^I-96kAID^-/- 5’-BE HTGTS libraries (n=3).

(h) Bar graph showing the percentage of junctions (average ± s.d.) recovered from the ΔSμ^2xI/ΔSγ1^2xI 3’-BE libraries (n=4).

(i) Bar graph showing the percentage of junctions (average ± s.d.) recovered from the ΔSμ^2xI /ΔSγ^2xI 5’-BE libraries (n=3).

(j) Bar graph showing the percentage of junctions (average ± s.d.) recovered from the WT ΔSγ1^2xI-3’BE libraries (n=3).

(k) Bar graph showing the percentage of junctions (average ± s.d.) recovered from the ΔSα^1xI

CH12F3 3’-BE libraries (n=3) and ΔSα^1xI Sμ^(INV) CH12F3 cells 3’-BE libraries (n=3).

(l) Bar graphs depicting percentage of trans junctions mapping to Sμ in (-) and (+) orientation from libraries of ΔSμ^2xI/ΔSγ1^2xI B cells (n=3) cloning from ΔSγ1^2xI 3’BE.

(m) Bar graphs depicting percentage of trans junctions mapping to Sμ in (-) and (+) orientation and to Sε in (-) and (+) orientation from libraries of c-myc^25x-I-sceI 5′BE (n=3).

(n, o) HTGTS libraries analyses of ΔSα^1xI CH12F3 cells stimulated with αCD40, IL4 and TGFβ and nucleofected with I-Sce1 expression plasmid. Cells were harvested on day 3 post-stimulation for 3’-BE (n,n=6) and 5’-BE (o, n=6) libraries.

(p) 3’-BE and 5’-BE libraries are normalized with “symmetric junctions” (see Supplementary Information).

(q) Bar graph showing percentage of junctions from ΔSα^1xI CH12F3 cells (n=6) from 3’BE or 5’BE primer.

For detailed legends refer to Supplementary Information.