Treatment of coverslips with RNases can selectively remove RNA and RNA–DNA hybrids. (A) Products from enzymatic reactions of oligonucleotide substrates with RNase III, incubated in commercially sourced ShortCut RNase III buffer supplemented with manganese. Hybrid, RNA–DNA hybrid. Products were resolved on a polyacrylamide gel and stained with SYBR Gold. (B) Same as in A, but oligonucleotides were digested with RNase T1 (T1); RNase III (III); RNases T1 and III combined (T1+ III); and RNases T1, III, and H combined (T1 + III + H) and incubated in low-salt, magnesium-containing buffer. (C) Representative confocal images of HeLa cells transfected with ATTO-594 (red)-labeled dsRNA. After fixation, coverslips were either mock treated or treated with a combination of RNases T1 and III. (D) Same as in C, but HeLa cells were transfected with ATTO-594 (red)-labeled RNA–DNA hybrids. After fixation, coverslips were treated with the following enzymes: none (No enzyme); RNase H; RNase T1 and RNase III combined (RNase T1 + III); or RNase H, RNase T1, and RNase III combined (T1 + III + H). S9.6 signal is shown in green and DAPI in blue. Scale bars are 10 microns; 5 microns for inset images. Inset images are magnified to show overlap of S9.6 signal and ATTO-594 foci. Bp, DNA size in base pairs.