shortcut rnaseiii  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: Synergistic action of human RNaseH2 and the RNA helicase-nuclease DDX3X in processing R-loops

doi: 10.1093/nar/gkae731

Figure Lengend Snippet: Silencing of DDX3X in human cells causes accumulation of genomic R-loops and phosphorylated RPA foci. ( A ) Representative immunofluorescence images obtained using antibodies against RNA:DNA hybrids (S9.6, red) and pRPA (green) after transfection of U2OS cells with control (si-control), DDX3X (siDDX3X), RNaseH2 (siRNaseH2A + siRNAseH2B) or DDX3X and RNaseH2 (siDDX3X + siRNaseH2A + siRNaseH2B) siRNAs. Treatment with RNaseH + RNaseIII + RNaseT1 was used to exclude unspecific S9.6 signals. Scale bars, 10 μm. ( B ) Quantification of mean nuclear intensity of phospho-RPA and S9.6 foci of control and DDX3X, RNaseH2A + B and DDX3x + RNaseH2A + B-depleted U2OS cells. Median with interquartile range (black lines) are indicated. P values were calculated using two-tailed unpaired Student's t test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant ( P < 0.05). At least 300 nuclei from three different experiments were analyzed. ( C ) Quantification of RNA:DNA hybrid Dot-blot of genomic DNA ± RNase H for all the siRNA conditions. S9.6 signals are normalized to dsDNA signal. P values were calculated using two-tailed unpaired Student's t test (means ± SEM; n ≥ 2).

Article Snippet: Enzymatic treatments were done in staining buffer supplemented with 3 mM magnesium chloride with 1:200 dilutions of RNaseT1 (EN0541; ThermoFisher Scientific), RNaseIII (AM2290; ThermoFisher Scientific) and RNaseH (M0297; NEB) and incubated for 1 h at room temperature.

Techniques: Immunofluorescence, Transfection, Control, Two Tailed Test, Dot Blot

Journal: Nucleic Acids Research

Article Title: Synergistic action of human RNaseH2 and the RNA helicase-nuclease DDX3X in processing R-loops

doi: 10.1093/nar/gkae731

Figure Lengend Snippet: Silencing of DDX3X in human cells causes accumulation of genomic R-loops and phosphorylated RPA foci. ( A ) Representative immunofluorescence images obtained using antibodies against RNA:DNA hybrids (S9.6, red) and pRPA (green) after transfection of U2OS cells with control (si-control), DDX3X (siDDX3X), RNaseH2 (siRNaseH2A + siRNAseH2B) or DDX3X and RNaseH2 (siDDX3X + siRNaseH2A + siRNaseH2B) siRNAs. Treatment with RNaseH + RNaseIII + RNaseT1 was used to exclude unspecific S9.6 signals. Scale bars, 10 μm. ( B ) Quantification of mean nuclear intensity of phospho-RPA and S9.6 foci of control and DDX3X, RNaseH2A + B and DDX3x + RNaseH2A + B-depleted U2OS cells. Median with interquartile range (black lines) are indicated. P values were calculated using two-tailed unpaired Student's t test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant ( P < 0.05). At least 300 nuclei from three different experiments were analyzed. ( C ) Quantification of RNA:DNA hybrid Dot-blot of genomic DNA ± RNase H for all the siRNA conditions. S9.6 signals are normalized to dsDNA signal. P values were calculated using two-tailed unpaired Student's t test (means ± SEM; n ≥ 2).

Article Snippet: We used 1U of RNaseIII (AM2290; ThermoFisher Scientific) per μg of genomic DNA for 2 h at 37°C and 10 U of RNaseH (M0297; NEB) per μg of DNA O/N at 37°C.

Techniques: Immunofluorescence, Transfection, Control, Two Tailed Test, Dot Blot