I'm making RNA fragments for RNA sequencing with my RNase III RNA Fragmentation kit. I'm having problems with fragment sizes. I need sizes of 350bp max and 100bp minimum, but My fragments are stuck at about 1000bp (even with 1 unit RNase, 37 degrees and I hour reaction!). I've observed some other experiments using fragmentation buffers without RNase and higher temperatures (80, 90 degrees centigrade). But I cannot do such because of my enzyme sensitivity. Also, the follow-up experiment (library making) requires the hydroxyl group tailing at the end of the fragments (as a result of the RNase cleaving).
So, I need suggestions. Do I keep increasing the reaction time and the enzyme units? (I've never seen anyone use above 1 hour though).
1000 bp RNA in bacteria sounds like a full length mRNA. Have you checked the activity of the enzyme from the kit? Maybe you were just working with a bad kit.
Alternatively, you can try sonication too,
I can also recommend sonication:
http://covarisinc.com/applications/dnarna-shearing-for-ngs/
If you need free cis-glycol groups at the 3'-end of RNA fragments, try several nucleases and find optimal conditions for cleaving. Those are: P1 nuclease, S1 nuclease or Mung bean nuclease. Good luck!
P.S. Sonication will not garantee free 3'-end hydroxy groups, then you will need phosphatase or S1 nuclese (mild conditions) additional treatment.
Yuri
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