RNase H-based cleavage assay to monitor 2′-O-methylation

The RNase H-based cleavage assay was performed as previously (24,52) with minor modifications. 250 ng of total RNA was denatured at 85°C for 3 min along with 1 pmol of chimeric 2′-O ribose methylated RNA-DNA oligonucleotide (Supplementary Table S6) in1X RNase H buffer (NEB) buffer. Samples were incubated on ice for 5 min, then mixed with 5 U of RNase H and incubated at 37°C for 30 min. Reactions were stopped by addition of 300 mM NaOAc pH 5.2 and 1 mM EDTA, and RNAs were extracted using PCI (25:24:1). The cleavage reaction products were resolved by denaturing agarose gel electrophoresis for monitoring sites on the 18S and 28S rRNAs or denaturing polyacrylamide gel electrophoresis in case of analysing sites on snRNAs. Cleavage products were detected by northern blotting using [³²P]-labelled DNA oligonucleotides probes as described above.