Chd2 depletion is independent of the capacity for self-renewal in undifferentiated mESCs. ( A ) Scheme of generating Chd2 mut/mut ESCs using the CRISPR/Cas9 system ( 25 ). The gRNA target site (chr. 7: 80, 664, 568–80, 664, 590) is indicated by a pink box. Details are shown in Supplementary Figure S1A . ( B ) Depletion of endogenous Chd2 protein in ESCs using the CRISPR/Cas9 system. Western blot analyses of WT and Chd2 mut/mut ESCs using antibodies against Chd2, Brg1, Oct3/4, Nanog, Sox2 and Hsp90 are shown. ( C ) Morphologies of WT and Chd2 mut/mut ESCs were not significantly changed under a bright field. Scale bars = 50 μm. ( D ) Cell cycle analysis of WT and Chd2 mut/mut ESCs by flow cytometry. The population of cells in each cell cycle phase (G 1 , S or G 2 /M) was calculated based on the intensity of Hoechst 33342. Data represent the mean of three independent experiments ± standard deviation. n.s., P > 0.05. ( E ) Scatter plot showing the expression levels of all genes in undifferentiated WT and Chd2 mut/mut ESCs. RNA-seq datasets were obtained and FPKM values were calculated. The red line indicates a regression line. ( F ) Venn diagram showing the overlap between genes with FPKM > 5 in WT and Chd2 mut/mut ESCs.