Cells were isolated form 8-10 week-old NOD mice and double sorted by flow cytometry for DAPI- CD4+ TCRb+ CD25hi (top 50%) Tregs 50,000 CD4+TCRβ+CD25hi (top 50%) Tregs were sorted and lysed. After transposition and PCR, a final bead purification and selection (100–600 bp) was performed twice using 0.6× and 1.6× SPRI beads. Libraries were paired-end sequenced using a 75-bp kit on an Illumina NextSeq high-throughput sequencing system.