cDNA circularization versus UDG based strand specific RNA-seq construction

[jazz]

I need to construct strand-specific RNA-seq libraries for a class of non-coding RNAs in my system and I am considering 2 alternative library construction methods for producing strand-specific reads on Illumina.
First method is based on using dUTP in second strand cDNA synthesis followed by UDGase mediated cleavage of that strand as per these
http://www.ncbi.nlm.nih.gov/pubmed/19620212 http://www.ncbi.nlm.nih.gov/pubmed/23273270

The other method involves circularization of cDNA using circligase by Ingolia et al.

http://www.ncbi.nlm.nih.gov/pubmed/19213877

Does anyone have any experience with either of these methods? Can someone comment as to which one might be better and why?

thanks

[jazz]

Just trying to bump this thread up. Anyone with experience in making strand-specific RNAseq libraries using the cDNA circularization method or 2nd strand synthesis with dUTP incorporation.

Thanks!

[nucacidhunter]

Is there any reason you are not trying to use one of commercially available strand specific RNAseq kits. Some have implemented a more efficient version of #1.

[jazz]

I am trying to prepare GRO-seq libraries and not mRNA-seq. Hence, I need to use a home-brew method versus a commercial kit.

[cmbetts]

Aren't most of the GRO-seq specific protocol bits around the isolation of the RNA, rather than library prep itself?

That being said, between those two choices, without commenting on actual library quality, dUTP based methods are currently the most popular, being implemented in most commercial kits. There's likely to have been more optimization and resources for help around dUTP.

[nucacidhunter]

I am trying to prepare GRO-seq libraries and not mRNA-seq. Hence, I need to use a home-brew method versus a commercial kit.

Comercial kits are not specificly for mRNA. The input can be any RNA including total, rRNA depleted, mRNA, etc. They use random primers for cDNA and dUTP for second strand synthesis. Insted of using UDGase, they use a polymerase that stalls at UTP positions, therefore only 1st strand is used for sequencing library.