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  • bpinder
    Junior Member
    • May 2013
    • 4

    Help with low input RNA-seq experiment design

    Hello,

    Newbie here trying to move forward with an RNA-seq project. Thank you for your patience.

    I have samples containing ~50ng of total RNA for RNA-seq. I am considering the SMARTER and OVATION technologies but have several questions regarding the workflow:

    1) When (and how) should I remove rRNA? I suspect it needs to be done prior to reverse transcription, but the kits I found online require more starting material.

    2) Do both kits support downstream barcoding for multiplexing?

    3) Would it be possible to instead make cDNA and perform Whole Genome Amplification followed by KAPA kit?

    Any other suggestions? I very much appreciate your feedback.

    Thank you,
    Ben

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  • GATTACAT
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    Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.
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  • SEQadmin2
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    I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.

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