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-   -   ScriptSeq v2 135base-peak (http://seqanswers.com/forums/showthread.php?t=86098)

JasperGeh 12-14-2018 04:22 AM

ScriptSeq v2 135base-peak
 
1 Attachment(s)
Hello people,

I tried the ScriptSeq v2 RNA Library Prep Kit on the total RNA extract of a cerebrospinal fluid sample. The RNA amount was not measureable with our Qubit but I tried anyway. I followed the protocol, did the AMPure bead purification, ran 16 PCR cyles and got this massive 135b-peak in the bioanalyzer that towers everything.
Any idea what this could be? I'm super new to RNA seq and not yet familiar with all the intricacies and pitfalls of the steps, so any help is appreciated :)

Cheers!

HESmith 12-14-2018 06:22 AM

Looks like adapter dimer.

nucacidhunter 12-15-2018 02:39 AM

Bead clean up with 0.8x should remove it. It may be necessary to do two clean ups for complete removal. Clean ups should be done with all samples that will be compared if the aim is DGE analysis.

JasperGeh 12-17-2018 02:15 AM

Quote:

Originally Posted by HESmith (Post 222055)
Looks like adapter dimer.

Alright, could be it. I assume there are a number of online resources on how to avoid dimer formation?

And thanks, I will try a 0.8x cleanup before sequencing.

luc 12-17-2018 11:31 PM

Randomprimer-dimers will be basically unavoidable for your low, unmeasurable input.
If the oligos are a separate reagent, you could try to reduce their concentration.


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