Figure 4. Single cell sequencing of inducible Bcl6-deletion in ILCs.

A) Diagram of experimental design for Cre activation on Id2^CreERT2 and Id2^CreERT2 x Bcl6^fl/fl mice. B) Representative flow cytometry plot (left) and quantification (right) of Id2^CreERT2 activation reflected by ROSA^tdRFP expression, on lymphocyte subsets from B) the mLN and C) SILP of Id2^CreERT2 mice following tamoxifen administration, n=3. D) Relative Bcl6 expression determined by RT-PCR on sort-purified tdRFP⁺ LTi-like ILC3 from the mLN of Id2^CreERT2 and Id2^CreERT2 x Bcl6^fl/fl mice, n=3. E) t-SNE and cluster identification of ILC single-cell RNAseq from the SILP of Id2^CreERT2 and Id2^CreERT2 x Bcl6^fl/fl mice. F) mRNA expression of subset-defining transcription factors and cytokines related to (E). G) LTi-like ILC3 sub-cluster identification (top) and genotype origin (bottom). H) Relative enrichment of LTi-like ILC3 sub-clusters on Id2^CreERT2 and Id2^CreERT2 x Bcl6^fl/fl mice, and I) relative genotype contribution to each LTi-like ILC3 sub-cluster as identified in (G). J) Dot-plot depicting frequency and normalised gene expression of selected genes across LTi-like ILC3 sub-clusters from (G). K) Il17a and Il17f mRNA expression on total ILCs from Id2^CreERT2 and Id2^CreERT2 x Bcl6^fl/fl mice, clustered as in (E). L) Volcano plot showing fold change (Log2FC) and adjusted P value (-log10(padj)) of regulon activity determined by SCENIC on LTi-like ILC3 from Id2^CreERT2 or Id2^CreERT2 x Bcl6^fl/fl mice. M) Regulon specificity score for each LTi-like ILC3 sub-cluster as identified in (G). Red labels indicate top most specific regulons in each cluster. Data representative of 2-5 independent experiments (B-D). Data represented as individual animals and mean. See also Figure S4 and Figure S6.