Computational protocol: DICER, DROSHA and DNA damage response RNAs are necessary for the secondary recruitment of DNA damage response factors

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[…] Cells were plated on coverslips or in glass-bottom dishes Mattek (P35G-1.5-14-C). Staining for NBS1, MDC1, 53BP1, γH2AX and BrdU were performed as described previously (). Cells were fixed in 4% paraformaldehyde followed by a 0.2% Triton X-100 permeabilization step if expressing a GFP or a YFP reporter gene or methanol:acetone 1:1 in all other cases. Coverslips and glass-bottom dishes were incubated for 1 h in 1× PBG blocking buffer [0.5% BSA, 0.2% gelatin from cold-water fish skin (Sigma-Aldrich, G7765) in 1× PBS]. Incubation with primary antibody, diluted in 1× PBG blocking buffer, was for 1 h at room temperature. Coverslips were washed three times with 1× PBG buffer and incubated with secondary antibody diluted in 1× PBG blocking buffer for 40 min at room temperature. Coverslips and glass-bottom dishes were extensively washed two times in 1× PBG blocking buffer and two times in 1× PBS. Nuclei were stained with 0.2 µg/ml DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich, D9564) in 1× PBS for 1 min at room temperature and coverslips were mounted in 1,4-Diazabicyclo[2.2.2]octane and incubated at room temperature overnight in the dark. After DAPI staining, glass-bottom dishes were incubated for 5 min with 4% PFA, washed and kept in PBS at 4°C. Images were acquired using a Leica TCS SP2 AOBS confocal system equipped with a Leica HCX PL APO 63×/1.4NA oil immersion objective and the Leica Confocal Software (LCS), keeping the Lookup Table (LUT) in a linear range using an oil immersion objective or with a widefield epifluorescence microscope (Olympus IX71) equipped with PlanApo 60×/1.40NA oil immersion objective, a Cool SNAP ES camera (Photometrics) and driven by MetaMorph software (Universal Imaging Corporation). Comparative immunofluorescence analyses were performed in parallel with identical acquisition parameters and imaging analyses were performed using ImageJ (NIH) and CellProfiler () software. […]

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