Leido MNase
28486547 /2017
MNase assay was performed using protocol described earlier with slight modification suitable for Leishmania donovani [37]. 2.5 ml of log phase cells (2x107 cells/ml) were centrifuged and resuspended in 500 μl of permeabilization solution-I (150 mM sucrose, 80 mM KCl, 35 mM HEPES pH 7.4, 5 mM K2HPO4, 5 mM MgCl2, 0.5 mM CaCl2) with 0.05% triton X-100. It was incubated at room temperature for 5 minutes. The cells were centrifuged and washed with permeabilization solution-I without triton X-100 and then resuspended in permeabilization solution-II (150 mM sucrose, 50 mM Tris-Cl pH 7.5, 50 mM NaCl, 2 mM CaCl2) containing micrococcal nuclease and incubated for 5 minutes at room temperature. To stop the enzymatic reaction, equal volume of 2X TNESK solution (20 mM Tris Cl pH 7.4, 0.2 M NaCl, 2 mM EDTA, 2% SDS, 0.2 mg/ml proteinase K) and lysis dilution buffer (150 mM NaCl, 5 mM EDTA) was added and incubated at 37°C in water bath for 2 hours. Isolation of MNase digested chromatin from highly permeabilized parasite was done using ethanol precipitation. Two volumes of cold ethanol and 3 M sodium acetate, pH 5.2 (0.3 M final concentration) were added and mixed by inverting and incubated at -20°C for overnight. It was centrifuged at 20,000g for 20 minutes at 4°C. The pellet was washed with 70% ethanol and air dried. The chromatin was dissolved in TE buffer (pH 8.0).
