Dnapac pa200 rs column

Ten microliters of the quenched reaction were automatically injected onto an analytical DNAPac PA200 RS column (4.6 × 250 mM, Dionex). Capped and decapped RNA were separated by anion exchange HPLC at 50°C using appropriate gradients (buffer F: 25 mM Tris, pH 8; buffer G: as buffer F, plus 1.5 M NaCl) at a flow rate of 0.45 ml/min. Eluting RNA was detected using the absorption at 260 nm (Supplementary Figure S1A).

Decapping assays were performed at 30°C in GF-buffer supplemented with 5 mM MgCl₂ and 0.005% Triton X-100 using 20 µM capped 21mer, 100 nM enzyme and 3 µM Edc constructs or 350 µM Xrn1 (1224–1257) or Dcp2 (267–350) peptide. Samples were taken at the indicated times, diluted one-eighth in 10 mM EDTA to stop the decapping reaction and chloroform/phenol extracted to remove the protein. Ten microliters of the RNA solution were analyzed by anion exchange HPLC chromatography at 50°C using a DNAPac PA200 RS column (Dionex; 4.6 × 250 mm) equipped with a guard column (Buffer A: 20 mM Tris, pH 8.0, Buffer B: 20 mM Tris, pH 8.0, 1.5 M NaCl, gradient 41%–50% B over 16 min, flow rate 0.45 mL/min). Elution of capped and decapped RNA was monitored at 260 nm, peak areas were integrated and corrected for different absorption coefficients of capped and decapped RNA to calculate the fractions of capped and decapped RNA species in the sample.