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  • mRNA fragmentation for SPRIworks

    We are making ds-cDNA for automated mRNA-seq library preparation using the SPRIworks robot. After the poly-A selection, our protocol calls for fragmentation at 80 degrees for five minutes using ambion reagents. We get a nice bump on the bioanalyzer when running the ds-cDNA, however the peak is around 400-500 bp (see attached traces). Using the standard illumina protocol, fragmenting for 2 minutes at 94 degrees, results in a broader around 150 bp.

    We plan to use the SPRIworks size selection of 200-400 bp. I am worried that after adapter ligation, much of our sample will fall outside of this range.

    Any opinions? Should I just leave it alone?

    We need to get some samples submitted to the robot by tomorrow, so I am planning to add 90 seconds onto the 5 minute fragmentation time. Is this a bad idea?
    5 minute, 80 degree fragmentation:
    [ATTACH]Click image for larger version

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    2 minute, 94 degree, illumina kit fragmentation:
    Attached Files
    Last edited by jowenMIT; 01-27-2011, 02:23 PM.

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