Hi all,
I have been doing some mRNA TruSeq sample prep, using the protocol *exactly* as advised by the Illumina folk (using PolyT beads to get mRNA, fragmenting for 7 minutes chemically, 15 PCR cycles), and I am getting an odd and consistent pattern where I have my desired peak ~300 bp and a second peak ~900 bp. See an exemplar trace attached.
Reading through the other posts, seems like most people think this due to overamplification... could that be my issue here? My biggest concern is (because the double peak is similar in size ratio to the rRNA size ratio) that this reflects rRNA contamination. Does anyone have experience with that?
Thanks!
I have been doing some mRNA TruSeq sample prep, using the protocol *exactly* as advised by the Illumina folk (using PolyT beads to get mRNA, fragmenting for 7 minutes chemically, 15 PCR cycles), and I am getting an odd and consistent pattern where I have my desired peak ~300 bp and a second peak ~900 bp. See an exemplar trace attached.
Reading through the other posts, seems like most people think this due to overamplification... could that be my issue here? My biggest concern is (because the double peak is similar in size ratio to the rRNA size ratio) that this reflects rRNA contamination. Does anyone have experience with that?
Thanks!
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