RNA Detection

2. Perform PCR on a thermal cycler using the following program
   parameters: 95C for 5 min; 25 cycles [denaturation 95C for
   30 s, hybridization 55C for 30 s, elongation 72C for 4 min
   (18S) or 7 min (25S)]; 72C for 7 min. Cool down to 20C.


3. Optional: check PCR amplification using agarose gel (see
   Note 3).


4. Transfer your PCR reaction in a 1.5 mL RNase-free microcen-
   trifuge tube.


5. Adjust the volume of the reaction to 200μL with RNase-free
   water. Add 200μL of phenol–chloroform mix and proceed to
   extraction. After centrifugation for 10 min at 16,000 g,
   transfer the upper (aqueous) phase to a new 1.5 mL microcen-
   trifuge tube.


6. Precipitate PCR template by the addition of 1/10th volume
   3 M sodium acetate and three volumes of 96% ethanol. Mix by
   inverting the tube several times. Incubate for 15 min at 80 C
   and centrifuge for 15 min at 12,000–16,000gat 4C.


7. Remove the supernatant and wash the pellet with 75% ethanol.


8. Dry and dissolve pellet in 20μL of sterile water.

3.2 In Vitro Yeast
rRNA Transcription
and RNA Purification

1. In a 1.5 mL RNase-free microcentrifuge tube, settle the tran-
   scription reaction in a final volume of 50μL by mixing atroom
   temperaturethe components in the following order: 10μLof
   transcription buffer, 1μL of RNase inhibitor, 16μL of rNTP
   mix, 12.5 μL (18S) or 11.1 μL (25S) of DNA template
   (100 nM final), and 3μL of T7 RNA polymerase.


2. Incubate at 37C for 2 h (seeNote 4).


3. Digest the DNA template by addition of 1μL of DNase RQ1
   and incubation for 20 min at 37C.


4. Adjust the volume of the reaction to 200μL with sterile water
   and proceed to phenol–chloroform mix extraction followed by
   a chloroform extraction.


5. Precipitate the RNA transcript by addition of three volumes of
   96% ethanol in the presence of 20μL of ammonium acetate and
   1 μL of glycogen. Mix by inverting the tube several times.


6. Incubate for 15 min at 80 C and centrifuge for 15 min at
   12,000–16,000gat4C.


7. After centrifugation, the RNA pellet is washed with 80% etha-
   nol, dried, and dissolved in RNase-free water.


8. Purify transcripts from unincorporated nucleotides by gel fil-
   tration on a Quick Spin RNA column according to the manu-
   facturer’s instructions.


9. Quantify RNA using the UV spectrophotometer.

Quantification of 2^0 - O-Me by RiboMethSeq 35