Dig labeled dntps

Total RNA (2.5–10 µg) was denatured for 10 min at 65°C before separation on a 1% formaldehyde gel (80 V, 6 h). After transfer to nylon membrane by capillary blotting (Membrane Hybond-XL, Amersham), the RNA was cross-linked with UV light. The RNA was visualized with 0.4% methylene blue and ladder position was determined. Hybridization was performed with a digoxigenin-labeled (DIG-labeled) dsDNA probe obtained through a PCR reaction with DIG-labeled dNTPs (Roche). Primers in the PCR reaction were Pro-FW1 and Pro-RW2, amplifying 333 nt, at the 5′-end upstream of the protospacer region, not covering the protospacer (Supplementary Table S3). The hybridization was done overnight at 42°C. Three stringent washing steps were made for 30 min each with 0.2% SSC/0.1% SDS at 55°C, 60°C and 65°C. Probe/mRNA hybrids were detected with DIG antibody (Roche) and chemiluminescence reaction with CSPD solution (Roche). Different amounts of total RNA were loaded for the blot shown in Figure 1B and Supplementary Figure S5 (∼2.5 µg for A53*, 10 µg for D63-7U and 5 µg for D63-HA) to allow visualization of the cleavage product of D63-7U.