Transciptional profiling of human mTEChi

Aire-sensitive genes exhibit a weak inclusion of alternative splicing events Thymi from cardiac surgery pediatric patients were minced in 1xPBS to release the thymocytes. Then treated four times with collagenase/dispase (1:200) and DNaseI (1:1000) digestion, and the cell fractions were further dissociated with gentleMACS treatment. All fractions were combined and filtered through the 100μm Falcon cell strainer. The cells were collected by centrifugation, washed, and transferred to Optiprep density gradient. The upper cell layer was combined into one tube, washed, and counted resulting in 0.4-6.5x 10^8 thymic cells. The cell fraction was then depleted with human CD45 beads (100ul beads per 2x10^8 cells; Miltenyi Biotec) using autoMACS. After the CD45 depletion, the cells were sorted with BD FACSAria for mTEC MHChi (CD45- CDR2- EpCAM+ HLA-DRhi) directly into TRIzol (ThermoFisher) solution.