Fig 1: scRNA-seq of Bmi1+ intestinal epithelial cells in response to diet: A, Rosa26tom marked Epcam+, CD45-negative epithelial cells FACs isolated from Bm1cre:er, Rosa26tom mice fed AIN76A or NWD1 for 3 months, then sacrificed at 3 or 66–70 days after TAM injection to activate the Rosatom marker (shorter, longer term, respectively) and analyzed by scRNA-seq (N = 2 for each group). B, Cell trajectory analysis as a function of diet and time after marking by TAM injection: Blue arrows, Ascl2 expressing cells among stem and progenitor cells; or yellow arrows, in goblet and enteroendocrine cells. C and D, Expression of Ascl2 per cell in Stem1, 2, Replicating and Dividing cell clusters: red arrow in D for Stem 2 cells is a population that expressed Ascl2 at a higher level. *** This was highly significant by a likelihood ratio test (P = 0.01), by a negative binomial regression-negative binomial mixed-effect model, with regression and fit using R functions glm.nb and glmer.nb, respectively (84), assuming each cell as independent, and number of Ascl2 reads for each cell as response. Furthermore, Ascl2 expression for NWD1-LT differed significantly from the other three groups (P = 0.002) by post hoc analysis by a similar negative binomial, and the potential that the difference between NWD1-LT and the other groups was random was P = 0.11, tested by a negative binomial mixed-effect model with mice per dietary group treated as random effects, indicating the alternate hypothesis that effects were random is false. E,Ascl2 and Bmi1 expression by ISH in mice fed diets for 3 months: white dotted lines demark the crypt base. For each of the four diet-timepoint groups analyzed in A–D,N = 2 mice per group.