Short-term R-loop disruption has minimal effects on mESCs. (A) Western blot of HA-RNASEH1 induction by doxycycline (Dox; 500 ng/mL) at 0, 24, 48, 72, and 96 h of induction. Tubulin is included as a loading control. (B) Disruption of R-loops by short-term induction of Rnaseh1. DRIP-qPCR is shown for several locations after 48 h of Dox treatment. Exogenous RNase H treatment after gDNA isolation is shown as an additional control. Mean and standard error of mean relative to the negative control locus are shown. (***) P < 0.001, (**) P < 0.01, (*) P < 0.05, (n.s.) nonsignificant (two-sided Student's t-test). (C) Changes in gene expression upon short-term Rnaseh1 induction in undifferentiated mESCs. Significance relative to log₂ (fold change) measured by RNA-seq (n = 3 replicates per condition) is plotted. Twenty-two downregulated genes (orange) and 11 upregulated genes (blue) (|log₂FC| ≥ 0.58, adjusted P-value ≤ 0.05) are indicated. (D) Gene expression of select genes from RNA-seq data. The expression level is transformed through regularized log transformation (rlog). Pou5f1 and Sox2 are genes with no significant changes; Rnaseh1, Hhip, Cdx1, and S100a1 are genes with significant changes upon Rnaseh1 induction.