Transcripts may have different and not yet annotated 3' ends, which might be mistaken for internal priming events of the oligo(dT) primer, when in fact those are true 3' ends. Artificial spike-in transcripts such as the SIRVs or the ERCC spike-in transcripts only have one defined 3' end, this which provides the only true measure to determine internal priming.
If true internal priming is detected, this could be a result of mis-priming during reverse transcription, for instance if the temperature before or during reverse transcription was too low. As outlined in the general section of the User Guide, unless
Unless explicitly mentioned, all steps in QuantSeq library preparation protocols should be carried out at room temperature between 20 °C and 25 °C.
In particularaddition, the temperature for First Strand cDNA Synthesis (p.11): is critical to ensure mis-priming of the oligo(dT) primer is avoided. Specifically:
Do not cool the FS2 / E1 mastermix (step 3)
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Have the RNA / FS1 samples at 42 °C when adding the FS2 / E1 mastermix (step 4) to avoid mishybridization.
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Do not forget to shortly spin down the samples at room temperature before and after adding the FS2 / E1 mastermix.
Mix samples properly by pipetting after adding the FS2/E1 mastermix.
ATTENTION: Centrifugation should not be carried out at 4 °C, always spin down at room temperature!
TIP! Raising the reaction temperature up to 50 °C for reverse transcription can be used for additional prevention of mis-priming. However, increasing the temperature to this level can reduce the efficiency of the reverse transcriptase enzyme, so this may result in a lower overall library yield.