Murine tracheas were dissected from 2 young (2-month) and 2 old (24-month) animals and dissociated to single cells in a two-step protocol. Tracheas were first incubated in papain and DNase  I for 30min at 37℃ with rotation, then triturated to release cells. Cells, debris and undissociated tracheal husks were pelleted and resuspended for a second enzymatic digestion in a mix of collagenase I, hyaluronidase, dispase, papain and DNase I for 20min at 37℃ with rotation. After the second digestion the husks including the cartilaginous scaffold dissociated fully. Single cells were filtered through a 100µm filter and red blood cells were lysed. Libraries were prepared using the Chromium Next GEM Single Cell 3' Kit v.3.1, according to the manufacturer's instructions.