RNA fragmentation

[miu007]

Hello all,

I am new to here and RNA-Seq. So I need some help.

I am working on optimizing the RNA fragmentation conditions with NEBNext RNA Fragmentation Buffer. I want to do 100 Paired end sequencing. Therefore I would like to generate 200-300bp fragments. However, I did not see any difference between incubating mRNA for 3mins and 5mins. The fragments are all less than 200bp, peaked at around 150bp. Is there anyone who also use this buffer for fragmentation? Can you share some tips on this? Much appreciated.

[kcchan]

Are you looking at the RNA size before or after the RNEasy cleanup? If it's after the cleanup it might be that your cleanup step is removing a lot of the longer fragments.

[miu007]

Thanks for your reply. The RNA was analyzed after cleanup. I used ethanol precipitation. It's supposed to retain all the fragments, right?