Click it edu detection

To assess cell cycle progression a dual thymidine labeling approach was used. EdU was administered through intraperitoneal injection to time pregnant females at 50 mg/kg followed by BrdU injection at 50 mg/kg 1.5 h later. 13.5 day old embryos were harvested 30 min after the BrdU injection, fixed in 4% PFA in PBS, brains frozen in 30% sucrose in PBS, and coronally cut at 12 μm on a cryomicrotome. Slides were incubated in 2 N HCl at 37 °C for 20 min and rinsed in PBS with 0.2% Triton X-100 prior to immunostaining with α-BrdU monoclonal antibodies (1:10; Life Technologies, Carlsbad, CA), α-Ki67 antibodies (1:200, Abcam, Cambridge, MA), and Click-iT EdU detection following the manufactures instructions (Life Technologies, Carlsbad, CA). Subsequently, mediolateral cortical aspects were imaged as described above and labeled cells of 200 μm wide cortical segments counted. Calculation of cell cycle parameters followed established paradigms^{40 (link),41 (link)}. Specifically, the duration of the experiment (T_I) was divided by ratios of single (EdU⁺) over double-labeled cells (EdU⁺/BrdU⁺) to generate estimates of S phase length (T_S). To estimate total cell cycle length (T_C), T_S was divided by the ratio of EdU⁺/BrdU⁺ cells over Ki67⁺ cells.

To quantify construct abundance in the screen for clonal expansion, head skin of mice at P21 was collected and digested in 0.25% collagenase at 37 C° for 1 hour to release mesenchymal cells from the epithelium. Head skins from two animals were pooled and treated as a biological replicate, to achieve a ~200 fold coverage. To quantify construct abundance in the renewal and cell division screens, head skin of mice at E18.5 was digested in 2mg/ml dispase at 37 C° for 1 hour to separate epidermis from dermis. Epidermal tissue was further digested with 0.25% trypsin for 30 minutes into single cells. Head skins from 4 animals were pooled together to make one biological replicate, thus achieving a ~45 fold coverage. For the renewal screen, single epidermal progenitor cells were stained with CD326/EpCAM-APC (G8.8, 1:50; BD Biosciences) and CD49f/α6-Integrin-PerCP (GoH3, 1:50; Biolegend). For the cell division screen, single epidermal cells were subjected for Click-iT EdU detection (Invitrogen, C10338) followed by CD49f/α6-Integrin-PerCP staining. Cell populations of interest were isolated using BD FACSAria II machine (BD Biosciences). gDNA from all samples was extracted using QIAamp DNA tissue mini kit (Qiagen). Barcode pre-amplification, sequencing, and data processing using Deseq2 program^{65 (link)} were performed as previously described^{27 (link),66 (link)}.