Rnase inhibitor cocktail

The cleavage assay was performed in 10 μL reactions containing 50 mM NaCl, 2.5 mM MgCl₂ and 20 mM Tris-HCl, pH 7.5, 5′-³²P-labeled substrate (10,000 cpm, approximately 5 nM) and 18 nM of the protein (hDcr or ∆DUF(625-752), or ∆DUF(630-709)). In addition, a reaction mixture without the protein was prepared as a control. In controls including EDTA, the reaction buffer was supplemented with the chelating agent to the final concentration of 25 mM. Reactions were carried out at 37 °C for 10, 30, 60 and 120 min with the addition of the commercial RNase-inhibitor cocktail (NEB, Ipswich, USA). The reactions were stopped by the addition of 1 volume of 7 M urea loading buffer and heating for 5 min at 95 °C. Samples were separated on a 15% denaturing polyacrylamide gel in 1 × TBE running buffer.

Cells were grown as described above. The cell pellet was washed with 10 ml of ice cold TMN buffer (10 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 100 mM NaCl) and resuspended in 940 μl of DEPC (Diethylpyrocarbonate)-treated ice cold water. Cells were permeabilized by the incubating with 60 μl 10% sarkosyl at 4 °C for 25 minutes with gentle shaking. Permeabilized ells were spun down by centrifugation at 1.2×g for 6 minutes at 4 °C. The cells were resuspended in 150 μl of transcription elongation reaction buffer (50 mM Tris-HCl pH 7.5, 100 mM KCl, 10 mM MgCl₂, 2 mM DTT, 0.75 mM each of ATP, CTP, GTP and Brd-UTP, 5 μl of RNAse inhibitor cocktail from NEB) and incubated at 30 °C for 5 minutes. Cells were immediately lysed by addition of 500 μl ice cold Trizol and 250 μl acid-washed glass beads followed by vigorous shaking at 4 °C for 20 minutes. The lysate was transferred to a new 1.5 ml microfuge tube containing 500 μl of Trizol and 200 μl of chloroform. The tubes were shaken vigorously and centrifuged at 14000 rpm at 25 °C for 20 minutes. The supernatant was extracted with phenol-chloroform three times. Total RNA was precipitated by incubating with NaCl overnight at −20 °C. The RNA pellet was washed once with ice cold 70% ethanol and resuspended in 100 μl of DEPC-treated water. RNA was further purified using the QIAGEN RNA Easy kit and eluted twice with 50 μl of DEPC treated water.

The cleavage assay was performed in 10 μL volumes in buffer containing 50 mM NaCl, 2.5 mM MgCl2 and 20 mM Tris-HCl, pH 7.5. The reaction mixture included ~5 nM ³²P-labeled substrate and dilutions of the protein preparation (12.5, 25, 50, 75 nM). In addition, a reaction mixture without protein was prepared as a control. In controls including EDTA, the reaction buffer was supplemented with the chelating agent to the final concentration of 25 mM. Reactions were carried out for 30 min at 37 °C with the addition of the commercial RNase-inhibitor cocktail (NEB, Ipswich, USA.). The reactions were stopped by the addition of 1 volume of 10 M urea loading buffer (10 M urea dissolved in water at 50 °C) and heating for 5 min at 95 °C. Samples were separated on a 15% denaturing polyacrylamide gel 1×TBE running buffer.