Thymic lobes from NMRI mice were collected and individually cultured on transwell inserts (0.4μm pores, polycarbonate membrane, Corning) at the air-liquid interface using complete medium (10% FBS) containing 1.35 mM 2-deoxyguanosin (2dGUO, Sigma). Six days later, lobes were intensely washed and cultured for 24h in hanging drops containing 50 000 DN3 (CD4-CD8-TCR−CD25+CD44-) cells FACS-sorted from thymocytes (CD8-depleted using mAb 31M and rabbit complement) from eight-week-old NOD or B6 mice. Lobes were then cultured on transwells for 12 days (cf. Fig. S1d). Lobes were collected and digested in RPMI containing 4μg/ml liberase and 0.1μg/ml DNAse I (Roche) during five minutes at 37°C with vigorous pipetting every two minutes. Cells were then washed, counted and stained for flow cytometry analysis.
Growth protocol
B6, BALB/c, C3H, CBA, DBA/1, and FVB mice were purchased from Janvier-labs, NOD and SJL from Charles River laboratories. Foxp3-Thy1a Rag2-Gfp-mutant B6 and NOD mice were previously described
Extracted molecule
genomic DNA
Extraction protocol
DNA was prepared from tail clips of three-day-old NOD, B6, F1, and BC1 mice using standard procedures
Label
Cy3, Cy5
Label protocol
DNA was prepared from tail clips of three-day-old NOD, B6 and BC1 (n=94) mice using standard procedures, quality-controlled using a fragment analyser (Agilent), quantified using PicoGreen
Hybridization protocol
hybridized to Illumina GGP Mouse GIGA-MUGA Arrays according to the supplier’s instructions (Infinium HTS Assay Protocol Guide, 15045738 Rev.A October 2013, Manual Protocol)
Scan protocol
iScan System (Illumina).
Description
Genomic DNA extracted from breast tumors was genotyped using Infinium HumanHap550-v3 Genotyping BeadChips (Illumina).
Data processing
The thus obtained raw-date were quality controlled and analysed using GenomeStudio Software 2010 with the Genotyping module (Illumina)
