Figure 1. Monoallelic Activation of V_κ.

(A) Single-cell RNA-seq Ig_κ expression profile of CD19⁺B220⁺CD43^loIgM^- small pre-B cells. Of 268 cells captured, number of cells expressing germline V_κJ_κ (blue), rearrangement in one Ig_κ allele (orange), rearrangement in more than one allele (purple), no V_κ or J_κ expression (green), or uncategorized (pink) are shown.

(B) Representative example of biallelic germline transcription of 5′J_κ1 promoter (magenta) without V_κ expression. mRNA splice products here and below are shown as arcs.

(C) Representative example of biallelic germline transcription of 5′J_κ1 promoter (magenta) with single V_κ expression from B6 allele (red).

(D) Representative example of biallelic germline transcription of 5′J_κ1 promoter (magenta) with multiple V_κ expression from CAST allele (blue).

(E) Percentage of cells with monoallelic expression as determined by either RNA-seq (left bars) or RNA-FISH (right bars). From left to right, percentages of cells expressing specific V_κ genes monoallelically (specific V_κ), cells expressing all V_κ genes from one allele (all V_κs), cells expressing highly used V_κs monoallelically (HUV_κs), and monoallelic J_κ expression (J_κ) are shown. Based on RNA-FISH, percentage of cells monoallelically expressing one or more assayed HUV_κs (green bar) is shown. Percentage of cells monoallelically expressing only one (1 HUV_κ, orange) or >1 HUV_κ (blue) is shown. Statistical significance was calculated on 100 cells per condition combined from two independent experiments by unpaired Student’s t test (***p < 0.001).

(F) Representative images of RNA-FISH for nascent transcripts from all nine highly used V_κ genes combined with staining for e-Pol II (blue). Distal V_κs are in green and proximal in red. The scale bars represent 1 μm.

(G) Heatmap of V_κs expressed from distal (V_κ 2–137 to 13–84), intermediate (V_κ 4–81 to 6–32), and proximal (V_κ 8–30 to 3–1) loops in each single cell (cell number given on right). Color reflects fraction of V_κ genes in indicated domains expressed in individual cells (scale 0%–25%).