I am going to perform circ RNA enrichment by RNAse R treatment and I have searched for the appropriate protocol but unfortunately did not find any detail. Most of them used different time interval ( from 10 min to 1 hour), reaction mixture content and phenol - chloroform extraction method after RNAse R treatment is not clear. I am going to use RNase R ( Epicentre). So if you have any idea, would you please help me ?
For RNaseR treatment, it depends on the quantity of input RNA. in their online protocol (http://www.epibio.com/docs/default-source/protocols/ribonuclease-r-e-coli.pdf?sfvrsn=8) they wrote that "One unit converts 1 μg of poly-r(A) into acid-soluble nucleotides in 10 minutes at 37°C in 20 mM Tris-HCl (pH 8.0), 100 mM KCl and 0.1 mM MgCl2.".
RNase R doesn't seem to be super heat stable in my hands, so I would try shorter times. 15m should be long enough, though some people do 30m or increase the temperature to 50C for more complete degradation. In these cases I would spike in more enzyme halfway through.
We have used 3 U RNaseR /µg total RNA for 15 min at 37°C for efficient depletion of linear RNA. Increasing to 10 U didn't result in further depletion while 1 U was not enough.
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