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Old 07-07-2018, 09:21 AM   #1
eaakimov
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Location: Helsinki

Join Date: Apr 2017
Posts: 1
Default RNASeq library generation protocol issue(like QuantSeq)

Hi,

I am trying to develop in-house protocol for RNA-seq library preparation in a way similar to Lexogen QuantSeq. QuantSeq includes double-stranded cDNA production with these major steps:
RT with dT(23)VN primer with overhang (i7 sequencing primer for NGS)
Removal of RNA (possibly with RNaseH)
Second strand synthesis with random primer with overhang (i5 seq primer for NGS)
These 3 steps are done without any purification. Then, they purify the product and perform PCR to add Illumina adapters.
I am wondering about step 3(second strand synthesis). I see the issue over here since dT(23)VN primer can also act as a primer for second strand synthesis. If so, during PCR to add Illumina adapters there could be an accumulation of a wrong product with two i7 adapters (adapters included in dT(23)VN primer). Any thoughts on how they overcome this issue? Thanks!
Short description for QuantSeq is here:
https://www.lexogen.com/wp-content/u...meth.f.376.pdf
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