Hi ALL
I am a young PI in Boston- working in Immunology-
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
1) using Nugen upstream for RNA prep of dUTP vs. subsequent Nugen multiplex kit vs. Illumnia kit
2) multiplexing with standard dUTP protocol- ie vest primers to use
Any help would be greatly appreciated.
I am a young PI in Boston- working in Immunology-
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
1) using Nugen upstream for RNA prep of dUTP vs. subsequent Nugen multiplex kit vs. Illumnia kit
2) multiplexing with standard dUTP protocol- ie vest primers to use
Any help would be greatly appreciated.