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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.

  1. 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.

  1. Add 42.5 μl freshly made bisulfite solution (EpiTect kit) and mix thoroughly by pipetting.

  2. Add 17.5 μl DNA stabilization solution (EpiTect kit) and mix thoroughly by pipetting.

  3. 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.

  1. 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.

  1. 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.

Ref. https://www.sciencedirect.com/science/article/pii/S0076687915002335?via%3Dihub

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    $\begingroup$ Did you read the manufacturer’s instructions? $\endgroup$ – canadianer Jul 7 at 17:43
  • $\begingroup$ yeah, but it is confusing for me. The Manufacture's link...bio-rad.com/featured/en/desalting-column.html $\endgroup$ – PraveenKumar Jul 7 at 17:55
  • $\begingroup$ That link does explain the basis of separation, and tells you the relative order of elution. You can take a look at the manual for more explicit directions. $\endgroup$ – canadianer Jul 7 at 22:06
  • $\begingroup$ Since it's a desalting column, without reading the instruction manual you could guess that the column will bind the RNA while you wash it with a new buffer. You will want what you elute at the end (RNA in elution buffer). The flowthrough will be the small salt molecules that wont bind to the column. $\endgroup$ – Cell Jul 7 at 22:40
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    $\begingroup$ @canadianer perhaps you should answer the question or if you think it is trivial (or needed more effort from the user, which is what I feel), you can VTC. $\endgroup$ – WYSIWYG Jul 9 at 11:59
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Maybe you can take a look at this. I found it through google when searching for the manual as well. http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006051.pdf

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