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Old 07-01-2013, 09:29 AM   #1
amarth
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Location: Mexico City

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Default ncRNA from RNAseq...

Dear all,

I have an experiment where I test three different conditions (you know, usual stuff ). Total RNA was extracted and sequenced by RNAseq, later determined gene expression for each condition, and so on...

Differential gene expression was made with bowtie,tophat,cufflinks,cuffdiff... and..

...One of my transcripts overexpressed was: ¡DICER! (a ribonuclease known to process pre-miRNAs and dsRNAs). I really suspect that my data from RNAseq has transcripts which are these non coding RNA, and I want to retrieve these sequences

The hard thing: How?

thx
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Old 07-01-2013, 10:23 AM   #2
NextGenSeq
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If you made your library using oligo-dT selection or priming you can't. Also, unless you isolated your RNA using Trizol or a kit such as Qiagen's miRNA-Easy kit you lost the small RNAs in the purification.
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Old 07-01-2013, 11:53 PM   #3
sphil
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If you don't lose them in sample prep one way is collecting all reads which didn't map to known coding regions etc. Assemble those into new transcripts (if you reach certain coverage) and look for ORFs inside your newly assembled transcripts to find the de-novo ncRNAs. The other workaround is to map and look especially for reads mapping to known ncRNA....
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