ScriptSeq - 2nd peak @ 550bp

[FerdinandvM]

Hi
I have been preparing libraries from approx 400ng total RNA, mRNA enrichment, using the ScriptSeqV2 kit.
At the end I amplify with 12 cycles and I get a profile with a 2nd peak around 550bp. I have not been able to get an explanation for this yet.

If somebody has an idea I would be grateful.

Thanks Ferdinand

P.S. Profile attached.

[Olaf Blue]

Did you quantify the input RNA for the Scriptseq library? The kit works best with 500 pg to 50 ng input.

[FerdinandvM]

No, but assuming that mRNA will never be more than 10% of the total RNA I should be in range.
Could it be an issue with the fragmentation?