Supplementary Figure 1: Validation of upregulated circRNAs in ISCs.

a, Validation of circRNA expression levels in Lgr5-GFP⁺ ISCs and Lgr5-GFP^– non-ISCs through real-time PCR. CircRNA expression levels were normalized to those of non-ISCs. n = 6 mice. b, Complementary DNA (cDNA) and genomic DNA (gDNA) were used as templates to amplify circRNAs in Lgr5-GFP⁺ ISCs with divergent and convergent primers. Due to large introns in gDNAs, different sets of divergent and convergent primers were used in the lower panel. c, Validation of circRNAs in Lgr5-GFP⁺ ISCs. Genomic compositions of circRNAs were depicted (upper panel). PCR products generated from Lgr5-GFP⁺ ISCs using divergent primers were sequenced (lower panel). d, Total RNAs from Lgr5-GFP⁺ ISCs were treated with or without 3 U/μg RNase R for 1 h, followed by RNA extraction and real-time PCR analysis. n = 3 independent experiments. e, Lgr5-GFP⁺ ISCs were treated with 2 μg/ml actinomycin D, followed by RNA extraction and real-time PCR analysis of circRNAs. n = 3 independent experiments. f, real-time PCR detection for knockdown efficiency of indicated circRNAs in Lgr5-GFP⁺ ISCs. n = 3 independent experiments. In all panels, data are shown as the mean ± s.d. ** P < 0.01; *** P < 0.001, by one-tailed Student’s t-test.