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Old 12-02-2011, 06:51 AM   #16
TonyBrooks
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Join Date: Jun 2009
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Quote:
Originally Posted by papori View Post
Maybe i just didnt understand what i read from this paper:
Development and applications of single-cell transcriptome analysis
Fuchou Tang1,2, Kaiqin Lao3 & M Azim Surani1

Combined with the existing strand-specific cDNA library preparation strategies, such as T7 RNA polymerase–based in vitro transcription and dUTP second-strand synthesis strategies it would be possible solve the stranded-ness of mRNAs is lost in the library construction, which prevents discrimination between sense and antisense transcripts from the same locus.

isnt it said that combined with both will solve the stranded-ness of mRNAs??
Can you explain that?

Thanks,
pap
OK. I've just read the paper. I think it's a bit confusing here as the paper is predominantly about single cell transcription profiling. In order to do this, you will need to amplify your starting material as there's no way you'll get enough library yield from one cell without amplification.

In fact, what the paper says is:

"Combined with the existing strand-specific cDNA library preparation strategies, such as T7 RNA polymerase–based in vitro transcription and dUTP second-strand synthesis strategies, it will be possible to recover the strandedness information for single-cell transcriptomes in the near future"

As I understand, it is implying that you could use T7-IVT to amplify into cRNA then from that create a cDNA library, with uracil incorporated into the second strand. This can then go into library prep, have the second strand degraded and finally PCR amplifed. It might give you enough material so to sequence whilst maintaining strandedness. Although IVT is a very 3' biased amplification, so there are probably going to be issues with this approach.
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