I have prepared libraries for RNA-seq using the Takara SMARTer Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian. The input RNA was extracted from FFPE tissue. Some of the final libraries have a shoulder or peak at around 160bp, as shown in the Bioanalyzer trace I have attached. I was wondering if anyone has had a similar issue, or could advise on the cause of this peak? Could it be due to adapter-dimers?
Many thanks,
Hattie
This is almost certainly adapter-dimer as the two adaptors are ~75bp so combined would total ~150bp. Most Illumina-based protocols cite that the final amplicon size of a library will be ~150bp larger than the shear/fragment size of the input DNA/cDNA. If this is before final AMPure cleanup, you should be fine following. If the peak is remaining, however, perhaps lower your AMPure bead to DNA ratio a little to preferentially recover larger fragments, or use a PippinPrep/TapeStation size-selection.
Hi Matthew,
Thanks very much for your answer. I took the library and performed an extra size selection with SPRIselect beads and a lower ratio of 0.8 which has seemed to work well.
Many thanks!
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