4 Notes
- Plasmid pHW18 was a generous gift from Dr. D. Tollervey. It
was used as a template to amplify yeast 18S and 25S rDNA. - The kit NEBNext®Multiplex Small RNA Library Prep Set for
Illumina®(set 1) (NEB, E7300S) includes a set of 12 barcod-
ing primers (numbered 1–12) that will be used for multiplexing
reactions during PCR amplification. There is also a version set
2 with primers (numbers 13–24). If you do not need these
barcoding primers, you may order a similar kit without the
primers and use any other source of barcoding primers (Illu-
mina, Epicentre, NEB). - After PCR, amplification may be checked by loading 1/10 of
the PCR reaction on a 1.5% agarose gel.
Fig. 2MethScore values for 2^0 - O-methylation sites in 18S and 25S rRNA for various proportions of unmodified
T7 rRNA transcript and naturally modified rRNA from yeast. Representative 2^0 - O-methylation sites were
selected and their calculated MethScores are traced according to proportion of modified and unmodified rRNA
sequence in the mix. Five points were measured: 100:0 (100% of in vitro rRNA transcript) to 0:100 (100% of
mature rRNA), with intermediate 75:25, 50:50, and 25:75 ratios. The right bottom panel shows the mean
MethScore values for all sites in 18S and 25S rRNA. Linear regression curves are also plotted. Correlation of
observed MethScore and methylation rate is>0.95 for all tested positions
Quantification of 2^0 - O-Me by RiboMethSeq 45