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Old 02-21-2011, 08:25 AM   #1
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Default RNA prep for Multiplexed- Strand Specific RNA seq


My lab is looking to do some RNA seq- and wants to look at both polyA and non-polyA RNA changes downstream of different cytokine signals.

While we will be looking at exon arrays, we are interested in also performing RNA-seq to look for novel transcripts, lcRNA, intron containing/pre-processed RNAs etc.

I am a total newbie to RNA seq- my former life was spent in the affy world...

I am looking to do some RNA seq- Want to analyze on Illumina HISeq- and to do some multiplexing to save money if possible- which I assume should be possible on the HiSeq instrument- We need strand specific sequencing- and presumably want paired end reads.

While I have lots of experience working with RNA for Affy/ Q-PCR etc.- I am a bit flummoxed by the variety of approaches for RNA prep for RNA seq- and want to look at both polyA and non-polyA RNA to broaden our discovery-

Based on the Levin paper (Nat Methods 2010)- I am assuming the best way to go is either dUTP or Illuminia kit- but I am wondering if anyone has any experience with other RNA preps prior to dUTP or Illuminia; specifically

1) using Nugen upstream for RNA prep for subsequent dUTP protocols?
2) copared Nugen vs dUTP directly? - My understanding of the Nugen multiplex label is that stand specificity is maintained in a similar manner to the Illuminia kits- no?
3) compared Nugen vs Illumina kits (I am rather fond of kits only for the relative ease of standardization across different lab members- have found in past life that so much of RNA work is dependent on the "hands" of the preparer- so want to minimize that influence)
4) multiplexing with standard dUTP protocol- ie best primers to use- any experience or papers out there?
5) Maximum number of samples one can practically multiplex on a single lane of HiSeq?- was planning on testing depth with multiplex of 4 vs. two just to see how much more we actually learn for the extra cost. Would be great if anyone out there has any real world experience from this sort of thing

Any help/advice would be greatly appreciated.
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