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).