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Old 01-27-2011, 02:21 PM   #1
jowenMIT
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Location: Cambridge

Join Date: Oct 2009
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Default 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]5-minute-80-degree-fragmentation.jpg[/ATTACH]

2 minute, 94 degree, illumina kit fragmentation:
Attached Images
File Type: jpg 2-minute-94-degree-illumina-kit.jpg (62.3 KB, 32 views)

Last edited by jowenMIT; 01-27-2011 at 02:23 PM.
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fragmentation, library preparation, mrna-seq, spriworks

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