RNA fragmentation using RNAse III: size issue

[CC_seqanswers]

I am trying to make RNA libraries for pair end sequencing(2x100). However I failed to get the RNA fragment size I need by using RNase III fragmentation. The average size of fragmented rRNA-depleted RNA is smaller than 200 nt even with half unit of RNAse III and incubation at 37C of 30seconds.

Any ideas to yield larger size?

[ZWB]

Try using NEBNext RNA fragmentation buffer. We do a 3 min incubation and get a pretty good size distribution for 100 PE runs

[CC_seqanswers]

Thank you! We have been quite succesful with NEB RNA fragmetation for pair end sequencing too.

It's just this time we are using Ambion kits and we need to use RNaseIII.

Try using NEBNext RNA fragmentation buffer. We do a 3 min incubation and get a pretty good size distribution for 100 PE runs

[pmiguel]

(1) I think you already under stand this, but you can't swap out RNAseIII digestion of RNA for heat/chemical fragmentation without altering your protocol! RNAseIII produces 5'-phosphate, 3'-hydroxyl ends -- exactly what RNA ligases need. heat/chemical fragmentation and most RNAses produce 3'-phosphate, 5'-hydroxyl ends.

(2) What is the average length of your RNA prior to RNAseIII digestion?

You might want to try an RNAseIII minus control reaction. That is, add all the reaction components, except RNAseIII (replace with water). Then do the normal incubation. If your RNA is still fragmenting, then it is contaminating RNAses in your RNA prep that are the problem, not the RNAseIII reaction.

--
Phillip

[CC_seqanswers]

Thanks for your help. Pls see replies below.

(1) I think you already under stand this, but you can't swap out RNAseIII digestion of RNA for heat/chemical fragmentation without altering your protocol! RNAseIII produces 5'-phosphate, 3'-hydroxyl ends -- exactly what RNA ligases need. heat/chemical fragmentation and most RNAses produce 3'-phosphate, 5'-hydroxyl ends.

[B]you are right.We are not swaping. RNAse III fragmentation is required for SOLID protocol, which is the same as Ambion.
[/B](2) What is the average length of your RNA prior to RNAseIII digestion?

You might want to try an RNAseIII minus control reaction. That is, add all the reaction components, except RNAseIII (replace with water). Then do the normal incubation. If your RNA is still fragmenting, then it is contaminating RNAses in your RNA prep that are the problem, not the RNAseIII reaction.

[B]It's hard to tell exact average length of the ribozero RNA samples but I would say around ~500bp? Ribozero RNA samples should be RNase free, however it does not hurt to a negative test. Had thought about it but forgot .--


B]
Phillip