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Figure S1. Gating strategy used for TEC sorting. (A) Mouse TEC-enriched cell suspensions were sorted as follows: PI-negative, CD45pos cells were sorted as thymocytes. PI-negative, CD45neg cells were separated into EpCAMposLy51high (cTECs) and EpCAMposLy51low (mTECs). mTECs were further separated into MHClow and MHChigh. (B) Human TEC-enriched cell suspensions were sorted as follows: DAPI-negative, CD45pos cells were sorted as thymocytes. DAPI-negative, CD45neg cells were separated into EpCAMposCDR2high (cTECs) and EpCAMposCDR2low (mTECs). mTECs were further separated into MHClow and MHChigh. (C) Mouse TEC-enriched cell suspensions derived from Adig mice were sorted as follows: PI-negative, CD45pos cells were sorted as thymocytes. PI-negative, CD45negEpCAMpos cells were separated into CD80lowGFPlow (CD80low), CD80highGFPlow (CD80highAireneg) and CD80highGFPhigh (CD80highAirepos).

Figure S2. Progressive structural disorganisation of Dicerdeficient mutant thymi. Thymic sections obtained from 4-, 10- and 14-week old Dicer null mutants and wildtype littermate controls were stained for cytokeratin-14, cytokeratin-8 and Aire. Epithelial voids are indicated with white asterisks. Note that the medulla disorganisation progressed with age, yet Airepos cells were still numerous in the Dicer mutants at 10 weeks. At 14 weeks of age, the Dicer null mutant thymus was very small and only a few cytokeratin-14-positive cells were detected. Aire staining is therefore not included for this age.

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