Fragmentation of RNA or cDNA

[Melanie]

Hi everyone,
I'm currently making some RNA-seq libraries from ribominus RNA (~500ng purified RNA per sample). I'm trying to work out what are the advantages/disadvantages of RNA versus cDNA fragmentation for a low starting sample.
Thanks!
Melanie

[kalu]

Hi Melanie
I have the same doubt... Did you sort it out?
Cheers, Kalu

[Melanie]

Hi Kalu,
From what I understand if you fragment the cDNA you get biases towards the 3' end of the transcript. If you fragment the RNA then you have a more equal distribution of reads from across the whole transcript with a relative depletion at both 3' and 5' ends. So it really depends what you are after.
Have a look at Wang et al. 2009 Nat Rev Genet 2009 vol. 10 (1) pp. 57-63 RNA-Seq: a revolutionary tool for transcriptomics

[kalu]

Hi Melanie
I synthesized full-lenght cDNA and amplified it using the SMART technology (SMARTer™ PCR cDNA Synthesis kit, Clontech). Do you think I would still be introducing a bias?
Many thanks.

[Melanie]

I'm sorry I wouldn't really know. I ended up going with RNA fragmentation in the end.

[NextGenSeq]

Yes, the SMART system gives the among the worst results according to the Broad paper

http://www.nature.com/nmeth/journal/...meth.1491.html