I want to perform the bisulfite sequencing on rRNA to detect the 5-mC. I followed this protocol
• DNA-free™ DNA Removal Kit (Invitrogen) • Purified RNA, Treat RNA according to manufacturer's instructions.
- Adjust DNAse-treated RNA to a volume of 10 μl.
2.Sodium Bisulfite Treatment of RNA EpiTect Kit (Qiagen)
• DNAse-treated RNA preparation • PCR thermal cycler • Micro Bio-spin 6 columns (BioRad) • GlycoBlue coprecipitant (LifeTechnologies) • Cooled microcentrifuge
3.Transfer DNAseI-treated RNA solution into a PCR tube.
Add 42.5 μl freshly made bisulfite solution (EpiTect kit) and mix thoroughly by pipetting.
Add 17.5 μl DNA stabilization solution (EpiTect kit) and mix thoroughly by pipetting.
Place samples in a thermal cycler and run the following program to deaminate RNAs:
• 70 °C: 5 min • 60 °C: 60 min • Repeat steps (i) and (ii) two to six times (depending on target RNA abundance and predicted secondary structures) • 25 °C—hold
7.Desalt reaction by passage through a Micro Bio-spin 6 column according to manufacturer's instructions.
- Add 1 volume (70 μl) of 1 M Tris–HCl (pH 9.0) and incubate at 37 °C—30 min for desulfonation.
9.Precipitate RNA by adding sodium acetate (pH 5.2) to 0.3 M, 15 μg GlycoBlue coprecipitant and 3 volumes of 100% ethanol and incubate at − 80 °C for ≥ 1 h.
10.Pellet RNA by centrifugation at 4 °C for ≥ 30 min at top speed.
11.Wash RNA pellet with 75% ethanol.
12.Air-dry ≤ 5 min at room temperature.
- Store deaminated RNA pellet until further use at − 80 °C.
In the step-7 I got stuck. i do not know whether do I have to use flowthrough or do i have to elute from the column.