% rRNA remaining after polyA selection

[jmRNA]

Does anyone have a standard or threshold of % rRNA they require in order to proceed with mRNA library prep?

In the past I've only proceeded with Illumina library prep after SeraMag polyA selection if I see 2% or less rRNA contamination on the bioanalyzer mRNA pico trace.

I'm currently using the Dynabeads polyA kit to obtain mRNA for ScriptSeq library prep. I've been getting anywhere from 0-7% rRNA with the same protocol (all samples had high RINs). I'm wondering if the 2% threshold is too stringent.

[mikaelk]

No I don't.
It depends a lot on your sequencing requirements.
If you do need a lot of coverage (so reads) on mRNA, keep the lowest thershold that you can.
If having a high coverage on transcripts is not of prime interest to you you ca be lazy on your threshold.
Without more information on what are your needs in terms of sequencing results, it is hard to say.

[pmiguel]

I think the %rRNA figure is going to be meaningless, or next to it, after polyA+ selection. This is because you will probably degrade your RNA a little during polyA+ selection, so any retained rRNA will no longer necessarily migrate where the bioanalyzer can recognize it as rRNA. If you want an accurate method, try qPCR.

Anything below 10% rRNA in your final sequence is probably going to be good enough. Actually up to even 50% is still usable. It is a waste of sequence reads, but the non-rRNA should still map, etc.

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Phillip