Figure S2.
Sort strategy, 10x Genomics workflow and validation.(A) FACS sort strategy to identify IgMhi and IgMlo TS B cell subsets. (B) Purity plots of sorted IgMhi and IgMlo TS B cell subsets. (C) 10x Genomics experimental workflow detailing pooling of HCD samples. (D) Summary table of cell numbers captured by the 10x controller and sequencing depth of IgMhi and IgMlo TS B cell subsets. (E) Violin plot demonstrating expression of the IL4R gene in IgMlo TS B cells. (F) Scatter plots of flow cytometry data demonstrating higher frequency of IL4R on IgMlo compared with IgMhi TS (CD27−IgD+CD10+) and naive (CD27−IgD+CD10−) cells (mean ± SD, paired t test). *, P < 0.05. (G) Violin plot demonstrating expression of the SELL (CD62L) gene in IgMlo TS B cells. (H) qPCR confirms higher levels of the SELL gene transcript in IgMhi TS B cells expressed as ΔCT values relative to an 18S endogenous control (paired t test). *, P < 0.05. (I) Ingenuity pathway analysis (IPA) upstream regulator plot demonstrating enrichment of LPS induced genes in IgMhi TS B cells. (J) IPA upstream regulator plot demonstrating enrichment of retinoic acid induced genes in IgMhi TS B cells. (K) IPA upstream regulator plot demonstrating enrichment of IFN-𝛾 induced genes in IgMlo TS B cells. (L) Bar graphs demonstrating a lower frequency of VH1 and higher frequency of VH3 immunoglobulin variable heavy chain gene usage in IgMhi TS B cells than TS IgMlo cells (Chi squared test with Bonferroni correction). *, P < 0.05; ***, P < 0.001. (M) Scatter plot of flow cytometry data from HCD demonstrating that T3 and naive CD45RBhi subsets as gated in Fig. 3 J share high IgM expression (mean fluorescence intensity [MFI] mean ± SD, paired t test). ****, P < 0.0001. (N) T3 and naive CD45RBhi subsets share similar high surface expression of CD24 (MFI mean ± SD, paired t test). ****, P < 0.0001. FSC-H, forward scatter height; SSC-H, side scatter height.

Sort strategy, 10x Genomics workflow and validation. (A) FACS sort strategy to identify IgMhi and IgMlo TS B cell subsets. (B) Purity plots of sorted IgMhi and IgMlo TS B cell subsets. (C) 10x Genomics experimental workflow detailing pooling of HCD samples. (D) Summary table of cell numbers captured by the 10x controller and sequencing depth of IgMhi and IgMlo TS B cell subsets. (E) Violin plot demonstrating expression of the IL4R gene in IgMlo TS B cells. (F) Scatter plots of flow cytometry data demonstrating higher frequency of IL4R on IgMlo compared with IgMhi TS (CD27−IgD+CD10+) and naive (CD27−IgD+CD10−) cells (mean ± SD, paired t test). *, P < 0.05. (G) Violin plot demonstrating expression of the SELL (CD62L) gene in IgMlo TS B cells. (H) qPCR confirms higher levels of the SELL gene transcript in IgMhi TS B cells expressed as ΔCT values relative to an 18S endogenous control (paired t test). *, P < 0.05. (I) Ingenuity pathway analysis (IPA) upstream regulator plot demonstrating enrichment of LPS induced genes in IgMhi TS B cells. (J) IPA upstream regulator plot demonstrating enrichment of retinoic acid induced genes in IgMhi TS B cells. (K) IPA upstream regulator plot demonstrating enrichment of IFN-𝛾 induced genes in IgMlo TS B cells. (L) Bar graphs demonstrating a lower frequency of VH1 and higher frequency of VH3 immunoglobulin variable heavy chain gene usage in IgMhi TS B cells than TS IgMlo cells (Chi squared test with Bonferroni correction). *, P < 0.05; ***, P < 0.001. (M) Scatter plot of flow cytometry data from HCD demonstrating that T3 and naive CD45RBhi subsets as gated in Fig. 3 J share high IgM expression (mean fluorescence intensity [MFI] mean ± SD, paired t test). ****, P < 0.0001. (N) T3 and naive CD45RBhi subsets share similar high surface expression of CD24 (MFI mean ± SD, paired t test). ****, P < 0.0001. FSC-H, forward scatter height; SSC-H, side scatter height.

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