Adp ribosyl cyclase

A 100-μL reaction containing 50 mM Hepes, pH 7.0, 5 mM MgCl₂, 45 μg cellular RNA, 10 μL of 3-azido-1-propanol (Sigma), 0.85 μM ADP ribosyl cyclase (Sigma), and 100 units of murine RNase inhibitor (NEB) was incubated for 30 min at 37 °C. The reaction was stopped by adding 100 μL phenol/chloroform (5:1, pH 4.5) (Invitrogen). The mixture was centrifuged at 12,000 × g for 5 min at 4 °C. The upper phase was collected and mixed with 100 μL chloroform. After centrifugation, the upper phase was gently collected and mixed at a 1:3 ratio with 100% ethanol and a 0.1× volume of 3 M NaOAc (pH 5.4). The RNA sample was incubated for 30 min at −20 °C and then centrifuged at 14,000 × g for 20 min at 4 °C. The pellet was washed twice with 400 μL 75% ethanol, air dried, and dissolved in 10 μL RNase-free H₂O. SPAAC was performed by incubating the RNA in a 30-μL reaction containing 155.2 mM NaCl, 2.97 mM Na₂HPO₄, 1.06 mM KH₂PO₄, pH 7.4, 30 units of murine RNase inhibitor (NEB), and 33.3 μM 39-nt-RNA-DBCO at 37 °C for 2 h. The RNA sample was mixed at a 1:3 ratio with ethanol and a 0.1× volume of 3 M NaOAc (pH 5.4). The RNA was pelleted by centrifugation at 14,000 × g for 20 min at 4 °C. The pellet was washed twice with 400 μL 75% ethanol, air dried, and dissolved in 40 μL RNase-free H₂O.