mRNA sequencing analysis of AID-TOM and REL-AID mouse spleen (SP) B cells after the second SRBC boost according to the expression of the tdTomato and YFP transgenes. (A) Schematic representation of the mouse immunization protocol. AID-TOM and REL-AID mice were submitted to a tamoxifen gavage at day D0, D2, D4 together with an SRBC immunization at d0. SRBC immunization boosts were done at M1 and M2. Animals were sacrificed 10 days after immunization. (B) Color code of the 5 CD19⁺ B-cell subsets sorted by fluorescence-activated cell sorter for RNA extraction and mRNAseq (see ”Materials and methods” and “Results”): TOM⁺ and TOM^– cells from AID-TOM mice, and TOM^–/YFP^–, TOM⁺/YFP^– and TOM⁺/YFP⁺ cells from REL-AID mice. In total, 30 B-cell samples from 14 mice were collected that were a priori classified as non–AID-imprinted (TOM^–, n = 6 and TOM^–/YFP^–, n = 8) and AID-imprinted B cells (TOM⁺, n = 6; TOM⁺/YFP^–, n = 3, and TOM⁺/YFP⁺, n = 7). (C) Color heatmaps for expression of tdTomato, YFP, and KOZAK-REL junction and for differential gene expression between TOM⁺/YFP⁺ and TOM⁺/YFP^– plus TOM⁺ B-cell samples. After unsupervised clustering of both genes and samples, 5 clusters of genes were identified, numbered C1 to C5 from the bottom to the top. On the left side, some key functions related to Gene Ontology annotations are shown for each cluster. On the right side are highlighted some key genes for each cluster. The number n of genes is given for each cluster in the heat map. Being immediately adjacent, the YFP and KOZAK-REL transgenes are in bold and underlined and are placed at their exact position in the clustering. (D) Frequency of the most abundant IgHV segment for each B-cell sample (ie, mRNA IgHV dominance). Mean and standard error of the mean are shown for each group of samples by a red and 2 black lines, respectively. ∗Mann Whitney P <.05; ∗∗∗Mann Whitney P <10^–3. mRNAseq, mRNA high-throughput sequencing. SD, standard deviation.