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Old 05-12-2010, 12:38 PM   #1
Melanie
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Location: New York, USA

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Default RNA-seq to detect expressed SNPs

Hi, I'm new to the sequencing world and have a few questions regarding a project I'm starting to look for expressed SNPs.

In order to maximise the number of SNPs sequenced I'm thinking about looking at pre-mRNAs, so currently I'm performing nuclear fractionation. My question is whether it would be better to do a ribosomal depletion (Invitrogen ribominus kit) or polyA selection? Has anyone had positive/negative experiences with these protocols and how easy is it to call SNPs from libraries prepared this way?

Any advice/comments would be greatly appreciated!

~Mel
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Old 05-13-2010, 07:36 AM   #2
krobison
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You might look at Illumina's new DSN protocol as yet another way to deplete total RNA of highly abundant RNAs such as rRNA.

It will be interesting to see how much more allele-specific transcription you can detect vs. existing papers which have looked for such using just mRNA
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Old 05-13-2010, 09:54 AM   #3
bioinfosm
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There was this recent webinar from Illumina (Schroth and Shujon) about rna-seq and they mentioned 3 approaches. If you can find that webinar archived. Here is same/similar that I found:
An Illumina-Demonstrated Method for Sequencing the Complete Transcriptome
https://illuminaevents.webex.com/ill...=a&d=663885452

sm
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