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  • Using diff of polyA and ribozero prepped RNA?

    Hi all,
    I'm wondering if anyone knows if there are advantages to having both polyA and ribozero prepped RNAseq data? For example, one could potentially use the diff to find non-coding RNAs?

    I'm preparing an experiment with yeast and am planning on duplicates for each timepoint. I thought it might be interesting to do one set of replicates with polyA-enrichment and one set of replicates with ribozero enrichment. Not only would I still get the statistical power of duplicates, I could also get a direct measurement on non-polyA, non-ribosomal RNA. Am I wrong?

    Thanks!

  • #2
    There will be many differences between these two procedures that may or may not have to do with the actual polyA status of the transcripts. polyA selection generates a strong 3' bias among transcripts, depending on the quality (RIN) of the input RNA. Reports at meetings recently have indicated that the correlation between polyA selected and ribo-depleted libraries from the same input total RNA can be as low as 0.9, depending on the method used for ribo-depletion. Therefore, this could confound your method.

    That said, I do think it could work, you just have to keep possible bias of the rRNA-depletion method in mind when conducting downstream analyses.

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