RNA fragmentation problem

[miu007]

Dear Mighty fellows,

I am new to RNA-Seq. Right now I am optimizing the RNA fragmentation conditions. I have tried incubating RNA with NEB Next fragmentation buffer for 3-5mins. All resulted in around 150nt fragments. I just tried 1min and 2min. In attached is a picture of that. My question is why there is always a 150nt peak in all conditions. Would anyone like to comment on that?

The other question is the fragment distribution after 2 min incubation too wide for RNA-Seq. I am making library for 100bp pair-end sequencing.

Your help is greatly appreciated.

[CottonJack]

Hi Miu,

I have just experience the same thing, fragmenting pure viral RNA. I`ve tried 6,7,8,9,10 min with this buffer and also different times (10, 15 min) with RNAse III module. Also, I always got the same size, around 200 bp.

Maybe decreasing (or increasing in my case, since I want smaller fragments) the concentrations would help.....