The cells were harvested, washed with phosphate-buffered saline (PBS) (Nacalai Tesque), and fixed in cold 70% ethanol for no less than 2 h. The cells were then washed and resuspended in 500 μL of assay buffer containing propidium iodide (PI) (Sigma-Aldrich) (20 μg/ml) and ribonuclease A (RNase A, Macherey–Nagel, Germany) (200 μg/ml) for 30 min at 20 °C in the dark. The DNA contents were analyzed using FACSVerse™ and FlowJo™ v10.8.1 (BD Biosciences, NJ, USA). A total of 10 000 events were conducted and examined as described previously59 (link).
Ribonuclease a
Manufactured by Macherey-Nagel
Sourced in Germany
Ribonuclease A is a common laboratory enzyme used for the digestion and degradation of ribonucleic acid (RNA). It is a small, single-chain protein that catalyzes the hydrolysis of the phosphodiester bonds in RNA molecules, resulting in the breakdown of RNA into smaller fragments.
Automatically generated - may contain errors
4 protocols using ribonuclease a
1
Cell Cycle Analysis by Flow Cytometry
2
Cell Cycle Analysis of Synchronized Fibroblasts
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Fibroblasts were cultivated on 100 mm by 20 mm tissue culture dishes (Falcon, catalog no. 353003) to ~70 to 80% confluence. For cell synchronization, cells were cultivated for 24 hours with DMEM/1% penicillin/streptomycin and 0.1% fetal calf serum. After 24 hours, the cells were harvested and prepared for cell cycle analyses. The cell pellets were resuspended in 500 μl of 70% ethanol and incubated at 4°C for 24 hours. The cells were centrifuged and resuspended in 200 μl of PBS with propidium iodide (50 μg/ml) (Sigma-Aldrich, catalog no. 25535-16-4) and ribonuclease A (25 μl/ml) (Macherey-Nagel, catalog no. 740505.50) and then incubated in the dark at RT for 30 min. The cell suspension was added to a fluorescence-activated cell sorting (FACS) Falcon and filled with 200 μl of PBS. Ten thousand cells were counted for routine FACS analyses (FACSCalibur).
3
Chromatin DNA Purification and Analysis
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Fifty-five point zero microliter of the total chromatin assembly reaction mixture was purified by phenol-chloroform-IAA (25:24:1, v/v) extraction (pH 8.0) followed by ethanol precipitation, and the purified DNA was resuspended in HD buffer (25 mM HEPES, 1 mM DTT, pH 7.6) containing a trace amount of Ribonuclease a (Macherey-Nagel GmbH & Co.). Supercoiled plasmid DNAs were separated by 0.8% TBE agarose gel in 0.5x TBE buffer, visualized with ethidium bromide (Nippon Gene), and analyzed by gel documentation system (Bio-Rad Laboratories).
4
Plasmid DNA Purification Protocol
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50 μL translation mixture was digested by 1 μL of 600 U·mL−1 Proteinase K (Wako, Osaka, Japan), and the plasmid DNA was purified by Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v·v−1, pH 8), followed by ethanol precipitation, and resolved in 6.5 μL HD buffer (25 mM HEPES, 1 mM DTT, pH 7.6) with the addition of a trace amount of Ribonuclease A (Macherey–Nagel GmbH & Co., Dueren, Germany). Samples were run on a 0.8% agarose gel in 0.5 × TBE buffer and stained by ethidium bromide. The gel image was analyzed using the ImageLab Software (Version 6.1.0 build 7, Biorad, Hercules, California).
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