Dneasy kit and protocol

Genomic DNA was extracted from isolates using the Qiagen DNeasy kit and protocol (Qiagen, Hilden, Germany). DNA libraries were prepared using Nextera XT (Illumina, San Diego, CA, USA) and whole genome sequencing was performed using the Illumina MiSeq with 2 × 300 bp chemistry. Small Molecular Real Time sequencing was performed on the RS-II (Pacific Biosciences, California, United States) using P6-C4 chemistry, and reference genome assembly was performed as described (Baines et al., 2016 (link)).

Splenocytes were harvested from 10-week-old WT and single TCR T cell mice. Erythrocytes were lysed using ACK buffer (0.15 M Ammonium chloride, 1 M potassium bicarbonate, 0.1 M EDTA in water) for 5 minutes at room temperature. Splenocytes were then enriched for T cells by using biotin conjugated anti-CD11b (BD Biosciences), anti-CD11c (eBiosciences), anti-B220 (eBiosciences), and anti-biotin magnetic beads (Miltenyi Biotec) before being passed through magnetic columns (Miltenyi Biotec). DNA was then purified from T cell-enriched cells using the DNeasy kit and protocol (Qiagen). Purified DNA was shipped to ImmunoSEQ (Seattle, WA) for TCRβ sequencing. Between 200,000 and 450,000 total sequences were obtained from each sample resulting in between 10,000 and 28,000 unique productively rearranged sequences per mouse. All ImmunoSEQ data are subject to quality control processing in efforts to minimize intrinsic amplification and sequencing errors [16 (link)]. The ImmunoSEQ data are contained in S1 Table.