Doxycycline

2 to 4 month old male and female K5^tTa;H2B^GFP mice were used (n≥3 mice per chase time point). To inhibit expression of the H2B^GFP allele, mice were initially given an intraperitoneal injection of doxycycline (0.4mg, Enzo Life Sciences, ALX-380–273-G005) and were then maintained on mouse chow that contained doxycycline (200mg/kg, BioServ, S3888). Mice were subsequently euthanized at 0 days (baseline), 3 days, 7 days, 21 days, 6 weeks, and 3 months after doxycycline treatment. The buccal mucosa in each mouse was removed, the epithelium separated from the underlying connective tissue, stained with DAPI in PBS for 30 minutes at room temperature, rinsed 2×5 minutes in PBS, and mounted onto glass slides as already described. Tissues were imaged in two ways. First, confocal microscope settings (e.g. laser power, exposure time, binning, gain, etc.) were adjusted to maximize the EGFP signal in the baseline K5^tTa;H2B^GFP samples (i.e. did not receive any doxycycline), without overexposing them and over-saturating the microscope camera. These same microscopic imaging parameters were then used to image the remaining K5^tTa;H2B^GFP samples in order to determine the relative differences in fluorescence between them. Next, optimized confocal settings were adjusted for each sample to maximize EGFP expression in order to detect any rare, label retaining cells at later chase time points.

For the short H2B–GFP pulse experiments, mice were injected once intraperitoneally with 200 μl of doxycycline (2 mg ml⁻¹, Enzo Life Sciences, ALX-380–273-G005) and then analysed at various timepoints after treatment (exact chase timepoints are provided in the main text). For the long pulse experiments (Supplementary Fig. 4f), mice were treated once with 200 μl of doxycycline (2 mg ml⁻¹) by intraperitoneal injection. Simultaneously, doxycycline was added continually to their drinking water (1 mg ml⁻¹ supplemented with 5% sucrose) for 3 weeks.