Sox2egfp

Mist1-CreERT mice [36 (link)] and Cxcl12-dsRED mice [37 (link)] were described previously. Cxcr4-EGFP mice were provided from Richard J. Miller (North-western University Medical School). LSL-Kras^G12D and LSL-Trp53^R172H mice were provided by Dr. Kenneth Olive (Columbia University). Cckbr-GFP BAC transgenic mice were purchased from MMRRC (GENSAT project [38 (link)]). Lgr5-DTR-GFP mice were provided by Genentech. Apc^flox/flox mice were obtained from the National Cancer Institute. Lgr5-EGFP-IRES-CreERT, Sox2-EGFP, R26-mTmG, R26-TdTomato, R26-LacZ, Cxcl12^flox/flox, and Tie2-Cre mice were purchased from the Jackson Laboratory. FucciG1 mice were obtained from RIKEN BRC. Cre recombinase was activated by oral administration of tamoxifen (TAM, Sigma, 3 mg/0.2 ml corn oil). All animal studies and procedures were approved by the ethics committees at Columbia University and the University of Tokyo. All mice were bred under specific pathogen free conditions. Comparisons were made with age- and sex- matched control animals.

Triple transgenic mice heterozygote for Fgfr3-iCreER^T2 (Young et al., 2010 (link)), Ai14:Rosa^tdTom (Jackson, #007908) and Sox2-EGFP (Jackson, #017592) were analyzed at P2, and double transgenic animals (Fgfr3-iCreER^T2 and Ai14:Rosa^tdTom) were analyzed at P21. Animals were injected with 0.2 mg/g body weight tamoxifen at P0 and P19 respectively. Animals were euthanized 2 days after the tamoxifen injection and organs of Corti were dissected. Single cell dissociation, flow cytometry, RNA isolation and single cell qRT-PCR were performed as described in Durruthy-Durruthy et al. (2014 (link)) using the primers listed in Table 2. Briefly, expression of Actb or Gapdh at levels lower or higher than 3 standard deviations from the mean was used to exclude compromised cells/empty wells or possible doublets, respectively. Ai14-Control primers detect recombination within the Ai14-tdTomato reporter locus and cells with no detectable recombination were excluded from the analysis. Single cell expression data is presented as Log2Ex values, calculated by subtracting experimentally determined Ct-values from the median limit of detection calculated for all primers used in the study. Single cell data were normalized using the median Log2Ex values as recommended by Fluidigm.