Mature mTEC were isolated and sorted according to their cell surface phenotype. Fragmented thymi were digested repeatedly for 15-20 min at 37 °C with 1mg/ml Collagenase/Dispase (Roche Diagnostic) and 100µg/ml DNaseI (Roche Diagnostic) in HBSS containing 2% FCS (Perbio), to obtain single cell suspensions. After the final digest, cells were pooled and labelled with biotinylated anti-EpCAM for positive enrichment by AutoMACS system (Miltenyi Biotec), and stained using the following directly labelled antibodies and reagents: FITC-anti-IAb (clone AF6-120.1, BioLegend), PE-anti-Ly51 (clone 6C3, BioLegend), PECy7-anti-CD45 (clone 30-F11, BioLegend), APC-Cy7-anti-I-A/E (clone M5/114.15.2, BioLegend), biotinylated anti-EpCAM (clone G8.8, DSHB, University of Iowa), Streptavidin-labelled PerCP-Cy5.5 (BioLegend) and Cy5-UEA1 (Vector Laboratories). The cells were exposed to 4’, 6-diamidino-2-phenylindole (DAPI) to identify dead cells and then sorted by flow cytometry (FACSAira, BD Biosciences) achieving a TEC purity of over 93%. Chromatin immunoprecipitation (ChIP) was performed as previously described (Adli M, Bernstein BE. 2011. Whole-genome chromatin profiling from limited numbers of cells using nano-ChIP-seq. Nat Protoc 6(10): 1656-1668) with the following modifications: protein G magnetic beads (Dynabeads, Life Technologies) were used to capture antibody-chromatin complexes. The captured complexes were washed four times with wash buffer (0.1 % SDS, 1 % Triton X-100, 0.1 % deoxycholate, 20 mM Tris- HCl (pH 8), 1 mM EDTA, 0.5 mM EGTA) containing 150 mM NaCl, once with wash buffer containing 500 mM NaCl, and finally once with LiCl wash buffer (250 mM LiCl, 1 % NP-40, 1% deoxycholate, 10 mM Tris- HCl (pH 8), 1 mM EDTA). Antibodies used were anti-H3K4me3 (ab8580, Abcam) and anti-H3K27me3 (#9733, Cell Signalling). ChIP-seq libraries were prepared with TruSeq ChIP Sample Preparation Kit (Illumina) and 50bp, paired-end reads were sequenced on an Illumina-HiSeq. For more information contact the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics, Oxford.