Episcript rt

A primer extension-based modification mapping method was used to analyze pseudouridylation of yeast and mouse U2 snRNA (Kiss and Jády 2004 (link); Deryusheva and Gall 2009 (link)). Fluorescently labeled oligonucleotides specific for vertebrate and S. cerevisiae U2 snRNA and for U87-based artificial substrate RNA were previously described ( Deryusheva and Gall 2013 (link); Deryusheva et al. 2012 (link)). To analyze S. pombe U2 snRNA, a new 6-FAM-labeled oligonucleotide was designed: [106–129] AAGCCAGAGGCTTTCCAACTCAAA. To detect pseudouridines, test RNA samples were treated first with CMC [N-cyclohexyl-N′-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate] (Sigma-Aldrich) for 20–30 min and then with pH 10.4 sodium carbonate buffer for 3–4 h at 37°C. Primer extension was performed using either AMV-RT (New England Biolabs) or EpiScript RT (Epicentre) at 0.5 mM dNTP concentration. Reaction products were purified by ethanol precipitation; dry pellets were dissolved in formamide, mixed with the GeneScan-500 LIZ Size Standard and separated on an ABI3730xl capillary electrophoresis instrument (Applied Biosystems).

To analyze 2′-O-methylation and pseudouridylation patterns of human U2 snRNA we used a fluorescent primer extension method as described (Deryusheva and Gall 2009 (link); Deryusheva et al. 2012 (link)). In brief, to detect 2′-O-methylated positions reverse transcription reactions were performed at very low concentration of dNTP. It has been shown previously that different reverse transcription enzymes have different termination efficiency at 2′-O-methylated positions (Deryusheva et al. 2012 (link)). We used EpiScript RT (Epicentre) to assess U2-Am30 semiquantitatively, and AMV-RT (New England Biolabs) to assess U2-Um47. To map pseudouridines, RNA samples were treated with CMC (N-cyclohexyl-N9-[2-morpholinoethyl] carbodiimide metho-p-toluene sulfonate) followed by incubation in sodium carbonate buffer (pH 10.4). Reverse transcription was done at 0.5 mM dNTP using EpiScript RT.
Each RNA sample was tested in two to three replicates. Sets of RNAs from parental and Nopp140KD lines were always treated simultaneously and separated on capillary columns in parallel using serial dilutions. GeneMapper 5 software (Applied Biosystems) was used to visualize and analyze the data.

Reverse transcription was performed using random hexamers with Episcript-RT (Epicentre) and AMV-RT (New England Biolabs) for 1 h at 37 °C. cDNA was amplified with inward and outward facing primers (SI Appendix, Table S1) using Taq DNA polymerase. RT-PCR experiments were performed in two independent replicates. To confirm the specificity of the amplified sequences, PCR fragments were cloned into the pGEM-T Easy vector (Promega) and sequenced.