Cells were fixed and prepared for indirect immunofluorescence and confocal microscopy as previously described11 ,26 (link).
Images were acquired on a Nikon Ti-E inverted microscope (Nikon) with a Yokogawa CSU-22 spinning disk confocal head with the Borealis modification or a Ti2 inverted microscope fitted with a CSU-W1 spinning disk. Z-stacks of 0.4–0.7 μm spacing were collected using a CoolSnap HQ2 CCD camera (Photometrics) or a Zyla 4.2 sCMOS camera (Andor) with a ×60/1.40 NA or a ×100/1.45 NA Plan Apochromat oil-immersion objective (Nikon).
The following antibodies were used for indirect immunofluorescence imaging: phospho γH2AX (Ser139) (Millipore, 05-636-I; 1:400); H3K27ac (Active Motif, 39133; 1:200); MDC1 (Abcam, ab11171; 1:1,000); MDC1 (Sigma-Aldrich, M2444; 1:1,000); phospho RNA PolII S5 (Millipore, MABE954, clone 1H4B6; 1:400); Cdk9 (Cell Signaling, 2316; 1:10); CDK12 (Abcam, ab246887; 1:400); 53BP1 (Santa Cruz, 22760S; 1:100); H3K27me3 (ThermoFisher Scientific, MA511198; 1:1,000); H3K9ac (Cell Signaling, 9649S; 1:400); H3K9me2 (Cell Signaling, 9753S; 1:400); POM121 (Proteintech, 15645-1-AP; 1:200); phospho H3T3 (Millipore, 07-424, 1:12,000); phospho H3S10 (Abcam, ab47297; 1:200); and fibrillarin (Abcam, ab4566; 1:500). Staining of Dam-methylated DNA in fixed cells was done using purified GFP-tagged m6A-Tracer protein as previously described53 (link).
Images were acquired on a Nikon Ti-E inverted microscope (Nikon) with a Yokogawa CSU-22 spinning disk confocal head with the Borealis modification or a Ti2 inverted microscope fitted with a CSU-W1 spinning disk. Z-stacks of 0.4–0.7 μm spacing were collected using a CoolSnap HQ2 CCD camera (Photometrics) or a Zyla 4.2 sCMOS camera (Andor) with a ×60/1.40 NA or a ×100/1.45 NA Plan Apochromat oil-immersion objective (Nikon).
The following antibodies were used for indirect immunofluorescence imaging: phospho γH2AX (Ser139) (Millipore, 05-636-I; 1:400); H3K27ac (Active Motif, 39133; 1:200); MDC1 (Abcam, ab11171; 1:1,000); MDC1 (Sigma-Aldrich, M2444; 1:1,000); phospho RNA PolII S5 (Millipore, MABE954, clone 1H4B6; 1:400); Cdk9 (Cell Signaling, 2316; 1:10); CDK12 (Abcam, ab246887; 1:400); 53BP1 (Santa Cruz, 22760S; 1:100); H3K27me3 (ThermoFisher Scientific, MA511198; 1:1,000); H3K9ac (Cell Signaling, 9649S; 1:400); H3K9me2 (Cell Signaling, 9753S; 1:400); POM121 (Proteintech, 15645-1-AP; 1:200); phospho H3T3 (Millipore, 07-424, 1:12,000); phospho H3S10 (Abcam, ab47297; 1:200); and fibrillarin (Abcam, ab4566; 1:500). Staining of Dam-methylated DNA in fixed cells was done using purified GFP-tagged m6A-Tracer protein as previously described53 (link).