Okay, I think you are getting ahead of yourself here. The simplest explanation for the result you posted initially is that your RNA is degraded. But it looks strange enough that it would be worth rerunning it. But for TruSeq DNAse treatment is recommended, so you might as well do that first -- if you clean everything up with a column. Then you can take a look at it on a chip again.
But, the most likely outcome is: degraded RNA, start again.
One step off the path as designed by Illumina, I'm willing to chip in suggestions. Two steps -- okay I'm still listening, not sure anything I say is going to be of much help. But you are 3 steps out now and on your own.
Let us know if it works, but you (1) have circumvented the normal QC step by declaring its failure was the result of genomic DNA in your sample -- which would not be there is you had not (2)also skipped a step asked for by the protocol by not DNAsing and now you (3)think that the fragmentation step should be sufficient to inactivate any DNAse still around after the polyA+ isolation. So, I can't say it will fail, but I don't like your chances.
The big problem is that the most likely failure mode, high 3' bias caused by low intactness of your total RNA, will not be obvious until you complete mapping back to a reference genome.
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Phillip
But, the most likely outcome is: degraded RNA, start again.
One step off the path as designed by Illumina, I'm willing to chip in suggestions. Two steps -- okay I'm still listening, not sure anything I say is going to be of much help. But you are 3 steps out now and on your own.
Let us know if it works, but you (1) have circumvented the normal QC step by declaring its failure was the result of genomic DNA in your sample -- which would not be there is you had not (2)also skipped a step asked for by the protocol by not DNAsing and now you (3)think that the fragmentation step should be sufficient to inactivate any DNAse still around after the polyA+ isolation. So, I can't say it will fail, but I don't like your chances.
The big problem is that the most likely failure mode, high 3' bias caused by low intactness of your total RNA, will not be obvious until you complete mapping back to a reference genome.
--
Phillip
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