RNA Detection

upstream T7 RNA polymerase promoter (underlined in the

sequence) to perform T7 transcription.

18S forward primer:

50 - TAATACGACTCACTATAGGTATCTGGTTGATCCTG-

CCAGTAG-3^0.

18S reverse primer: 5^0 -TAATGATCCTTCCGCAGGTTC-3^0.

25S forward primer:

50 - TAATACGACTCACTATAGGGTTTGACCTCAAATCA-

GGTAGG-3^0.

25S reverse primer: 5^0 -ACAAATCAGACAACAAAGGC-3^0

2. 25 ng/μL plasmid DNA template pHW18 (seeNote 1).


3. 2.5 U/μL Pfu DNA polymerase.


4. 10Pfu DNA polymerase buffer.


5. dNTP mix: 1.25 mM each.


6. RNase-free water.


7. Individual RNase-free 0.2 mL PCR tubes.


8. PCR thermal cycler (e.g., Agilent SureCycler 8000).


9. RNAse-free 1.5 mL microcentrifuge tubes.


10. Phenol–chloroform mix (1:1, v/v).


11. 3 M Na-Acetate (NaOAc) in water, pH 5.2.


12. 96% ethanol.


13. 75% ethanol.


14. Tabletop centrifuge.

2.2 In Vitro Yeast
rDNA Transcription
and RNA Purification

2.2.1 In Vitro Yeast rDNA
Transcription

1. 5Transcription buffer: 400 mM HEPES–KOH, pH 7.5,
   120 mM MgCl 2 , 10 mM spermidine, 200 mM DTT.


2. 40 U/μL RiboLock RNase Inhibitor.


3. rNTP mix: 12.5 mM each.


4. 400 nM 18S and 450 nM 25S PCR templates.


5. 20 U/μL T7 RNA polymerase.


6. RNAse-free 1.5 mL microcentrifuge tubes.


7. 37C incubator.


8. 1 U/μL RQ1 RNase-free DNase.

2.2.2 Purification of In
Vitro Transcripts

1. RNase-free water.


2. Phenol–chloroform mix (1:1, v/v).


3. Chloroform.


4. 5 M ammonium acetate (NH 4 OAc).


5. 10 mg/mL glycogen coprecipitant.


6. Refrigerated tabletop centrifuge.


7. 96% ethanol.

Quantification of 2^0 - O-Me by RiboMethSeq 31