Rnaase a

Cells were cultured for 36–40 h before irradiation, and were harvested by trypsinization at 48 h following irradiation. The cell number was enumerated with the help of a cell hemocytometer. The extent of proliferation was assessed by calculating the proliferation index (Px) as follows:
Proliferation index Px = N_t / N₀;
where N_t is the cell number at different post-irradiation times and N₀ is the number at the time of plating. Cells were fixed with ice-cold 80% ethanol and stored at 4°C for at-least 24 h before analyzing the DNA content by flow cytometry. Ethanol fixed cells were washed twice with PBS and incubated with RNAase-A (cat# 10109142001, Sigma, USA) for 45 minutes at 37°C. Cellular DNA was stained with propidium iodide (25 µg/mL) (0.1 x 10⁶ cells/100µL of PBS) and incubated in dark at room temperature for 30 minutes. PI-stained cells were then subjected to flow cytometry with CytoflexS (Beckman Coulter, California, USA). All the data was acquired with CytExpert software and was provided with a cytometer. Acquired data were analyzed with the FlowJo_10.4 version. The percentages of cells in G0/G1-, S-, and G₂+M phases were determined, after filtering for doublets and aggregates using the inbuilt software.

Chromosomal DNA was isolated from liquid cultures of protosteloid amoebae. Adherent cells were washed in phosphate buffer and harvested by centrifugation at 200 x g which was sufficient to deplete nearly all residual yeast cells. Trophozoites were lysed in 50 mM Tris–HCl (pH 7.5) with 2% [w/v] of SDS and 0.5 mM Na₂EDTA, followed by a 1-h incubation at 60°C with RNAase A (Sigma–Aldrich, Taufkirchen, Germany) and Proteinase K (Sigma–Aldrich) at final concentrations of 100 and 200 µg ml⁻¹, respectively. Further purification steps like extractions with phenol-chloroform and precipitation with isopropanol were carried out as described (Sambrook and Russell 2001 ).
For the isolation of total RNA, cells were rapidly harvested from the agar surface, resuspended in 2 ml of phosphate buffer, and centrifuged for 1 min at 6,000×g. Pellets were shock-frozen in liquid N₂ and stored −80°C. RNA was prepared by phenol extraction with subsequent additions of 500 µl of TRIsure (Bioline, Luckenwalde, Germany) and 200 µl of chloroform. Phase separation was achieved by centrifugation followed by an additional extraction with 500 µl of chloroform. Precipitation was as for DNA. RNA quantity and impurities were determined spectrophotometrically. Integrity of the isolated RNA was checked by agarose gel electrophoresis