RiboMethSeq was essentially performed as previously reported (32 (link)). Briefly, 150 ng of total RNA was fragmented under denaturing conditions using an alkaline buffer (pH 9.4) to obtain an average size of 20 to 40 nucleotides. Fragments were end repaired and ligated to adapters using a NEBNext Small RNA kit for Illumina. Sequencing was performed on an Illumina HiSeq1000. Reads were mapped to the yeast rDNA and snoRNA sequences, and the MethScore (fraction methylated) was calculated as MethScore (for ±2 nucleotides) (93 (link)), equivalent to “ScoreC” in Birkedal et al. (31 (link)). Statistical significance was determined by Student’s t test (P < 0.05).
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Quantitative RNA Methylation Analysis
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Corresponding Organization :
Other organizations : Emory University, Inserm, Centre National de la Recherche Scientifique, Université de Lorraine
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Variable analysis
independent variables
- Fragmentation of 150 ng of total RNA under denaturing conditions using an alkaline buffer (pH 9.4) to obtain an average size of 20 to 40 nucleotides
- MethScore (fraction methylated) calculated as MethScore (for ±2 nucleotides)
- Statistical significance determined by Student's t test (P < 0.05)
- Sequencing performed on an Illumina HiSeq1000
- Reads mapped to the yeast rDNA and snoRNA sequences
- No positive or negative controls were explicitly mentioned in the input protocol.
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