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Read 2 generates sequences corresponding to the original RNA molecule. Please ensure the correct strand specificity in the corresponding parameters.
For sequencing details we refer to Appendix G: Why do I see a second peak in high molecular weight regions between 1, - 9, bp on the Bioanalyzer traces?
A second peak between 1,—9, bp Fig. The library prep has been very efficient and a lot of cDNA was generated. Hence, the PCR ran out of primers and template started to denature and reanneal improperly.
This results in longer, bulky molecules that migrate at a lower speed on the Bioanalyzer chip or gels. This can interfere with exact library quantification if relying solely on the Bioanalyzer results.
Overcycling may lead to a distortion in gene expression quantification and hence should be avoided. How many PCR cycles are needed to amplify the libraries? The reference values given in Appendix B, p.
To be on the safe side and prevent under- or overcycling of your sample we recommend doing a qPCR first. Conduct at least 35 cycles to make sure the amplification reaches the plateau. For the sample in Fig. If the optimal cycle number lies within two values, it is recommended to always round up to the higher number in order to get more yield.
Once the number of cycles for the endpoint PCR is established for one type of sample, you can use it in the following experiments.
Calculation of the number of cycles for the endpoint PCR What is a typical library fragment size? For highly degraded or FFPE samples the final library size is quite dependent on the level of degradation, but will be around bp.
Can the insert size be regulated? This is accomplished by modulating the insert range of the library generated after second strand synthesis by different size cut offs during magnetic bead purification.
Please consult Appendix D: For highly degraded or FFPE samples the final library size depends on the level of degradation and cannot easily be adjusted.
The insert size will be around bp and shorter sequencing lengths are recommended. How long are the sequences hybridizing to the RNA?
Should those sequences be trimmed? Therefore, the first nine nucleotides need to be removed from Read 1 and the first six nucleotides from Read 2. What barcoding indexing system do you use? SENSE libraries can be multiplexed.
Multiplexing QuantSeq libraries with other library types in the same sequencing lane is not recommended. For further information please see FAQ 1.Cadillac User Experience seamlessly connects you to information, communication and entertainment.
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