RNASeq library generation protocol issue(like QuantSeq)

[eaakimov]

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/uploads/2015/04/nmeth.f.376.pdf

[nucacidhunter]

It should not be an issue as it requires polyA region for binding and priming. If there are such sequences (polyT) throughout the exons then some 1st strand cDNA might be primed but it will have negligible effect as these will not be sequenced due to lack of both flow cell binding motives.

[lexogen]

3' mRNA-Seq

Thank you for your interest in QuantSeq!
If we can be of any help with your RNA-Seq library prep needs, please contact us at [email protected].

Please note that there is also a highly customizable version called QuantSeq-Flex.

Best regards,
Lukas Paul

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

[seqsuave]

Has anyone tried half-reactions for the QuantSeq chemistry?