RNA Detection

16. Chloroform.


17. 3 M NaOAc in water, pH 5.2.


18. 96% ethanol.


19. 80% ethanol.


20. Refrigerated tabletop centrifuge.

2.5 Yeast rRNA
Reconstruction

1. 18S and 25S in vitro transcripts.


2. 18S and 25S purified RNAs.


3. 3μg/μL yeast total RNA preparation.


4. 1.5 mL RNase-free microcentrifuge tubes.


5. RNase-free water.


6. Agilent 2100 Bioanalyzer.


7. Agilent RNA 6000 Pico kit (50–5000 pg/μL).


8. Chip priming station (Agilent).

2.6 Alkaline
Hydrolysis and RNA
Fragmentation Quality
Control

2.6.1 Alkaline Hydrolysis

1. Sodium bicarbonate buffer: 100 mM NaHCO 3 pH 9.2.


2. RNase-free water.


3. Individual RNase-free 0.2 mL PCR tubes.


4. PCR Thermal cycler (e.g., Agilent SureCycler 8000).


5. RNase-free 1.5 mL microcentrifuge tubes.


6. Refrigerate tabletop centrifuge.


7. 96% ethanol.


8. 80% ethanol.


9. 15 mg/mL GlycoBlue coprecipitant (e.g., Ambion).


10. 3 M NaOAc in water, pH 5.2.


11. Dewar containing liquid nitrogen.

2.6.2 RNA Fragmentation
Quality Control

1. Agilent 2100 Bioanalyzer (Agilent Technologies).


2. Agilent RNA 6000 Pico kit (quantitative range 50–5000 pg/μL).


3. Chip priming station (Agilent Technologies).


4. RNase-free 1.5 mL microcentrifuge tubes.

2.7 End-Repair 1. RNase-free water.

2. 5 U/μL Antarctic Phosphatase.


3. Antarctic Phosphatase Buffer 10.


4. 10 U/μL T4 PNK.


5. T4 PNK Buffer 10.


6. 10 mM ATP.


7. 40 U/μL RiboLock RNase Inhibitor.

Quantification of 2^0 - O-Me by RiboMethSeq 33