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Old 11-05-2010, 04:01 PM   #5
JMorlan
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Location: Redwood City, CA

Join Date: Oct 2010
Posts: 4
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We recently completed an RNA-seq study combining Illumina's RNA-Seq protocol and multiplex kit and running on the GAIIx. Our data quality was excellent in terms of total read yield, %Q30, % unique mapping, etc. The only caveat is that library yields are rather dramatically reduced (a few were right at 10 nM post-PCR) compared to the standard protocol but we always had more than enough to cluster.

Illumina has confirmed with us that the multiplex kit reduces library yields (hence the increase to 18 cycles in the protocol).

I would recommend that you quantitate rather precisely (qPCR or PicoGreen) as you may just have low-yield libraries.

Incidentally, we also use a "Home-Brew" version of Illumina's RNA-Seq kit, with reagents/oligos purchased from 3rd-party suppliers (the Illumina kits are a bit expensive) so I don't think it's a problem that you are using NEB kits.

-John
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