cDNA prep for Illumina using the Nextera kit

[fgoetz]

Hello!

We are looking to streamline our cDNA sample prep for the HiSeq and many of our samples have very low quantities of RNA and are very hard to collect. So far we have tried the Nugen Ovation V2 kit for cDNA synthesis and finished with the TruSeq kit. We are waiting to see what the data look like but the yields look great!

Has anyone tried working up cDNA with Nextera? If so, how did it go and do you have any advice on this?

Has anyone tried following the Nugen Ovation V2 with the Nextera kit? If so, I would appreciate hearing any experiences, advice or suggestions.

Thank you for your time!

[kmkocot]

I have the same question. I'm working with microscopic animals and making cDNA libraries using the Clontech SMARTer kit but it just doesn't yield enough ds cDNA for TrueSeq library prep.

I imagine that the Nextera kit would result in bias towards sequencing the middle regions of transcripts? Illumina's FAQs say that Nextera yields fragments between 300 and 1000 bp so I'm worried that some short transcripts might not get tagmented at all.

[kmkocot]

After doing a little more searching it looks like it can be done!

http://epicentral.blogspot.com/2010_08_01_archive.html

The Myers lab at the Hudson Alpha tested it and got good sequencing results with human mRNA.

Thanks,
Kevin

[kmkocot]

Just a bump...

Has anyone tried the new Nextera XT sample prep kit with cDNA? They say it's good for small genomes, plasmids, and amplicons but nothing is said about cDNA.

Thanks,
Kevin