Does anyone have experience with one of the following kits, used to do cDNA synthesis and strand specific sequencing?
http://www.clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/Next_Gen_Sequencing_Kits/Total_RNA-Seq/Strand-Specific_RNA_Seq_Illumina
- https://www.neb.com/products/e7420-nebnext-ultra-directional-rna-library-prep-kit-for-illumina
http://www.biooscientific.com/next-gen-sequencing/illumina-stranded-rna-seq-library-prep-kits/nextflex-rapid-directional-rna-seq-kit
I've found three companies that offer this kind of kit, but am wondering how good they work and if they really are better than cDNA synthesis and subsequent Nextera library prep?
Many thanks for your input!
Basically it is still cDNA synthesis, however the one strands is uridilated and can not participate in the following PCR. Illumina stranded kit works best in our hands. As a side note I would not do any RNAseq if I have a choice.
As Petre says, it is cDNA synthesis, with the difference being that uracil is added during second strand synthesis to mark one of the strands for later degradation.
I have used Bioo's Nextflex directional kit and it worked well for me, though I'm sure any of these kits would be fine. I think directional RNAseq is definitely the way to go as you see huge differences when comparing data from directional and non-directional libraries. This can make a big difference when comparing gene expression levels and splicing since you can exclude counting reads from the antisense strand that would otherwise bias your results.
We have also used the Illumina kit and it worked very well in our hands even if we used relatively small amounts of material (~100ng of total RNA). It worked as well as the kit for unstranded RNAseq.
I agree with Cameron: unless you have a specific reason why you don't want the strand information, stranded mRNAseq seems to be the way to go.
Here is my method:
Simultaneous generation of many RNA-seq libraries in a single reaction.
http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.3313.html
I have good experience with Alex Shishkin's method, mentioned above.
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