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Old 11-22-2011, 11:24 AM   #1
rrdavis
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Location: Sacramento

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Default Ribo-Zero and TruSeq incorporation

Hi,

Does anyone have a protocol to use the ribo-zero kit in conjunction with the TruSeq reagents. We have some compromised RNA samples and would like to use both the TruSeq and Ribo-Zero kits from Illumina.

Thanks!

Ryan
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Old 11-23-2011, 07:05 AM   #2
pmiguel
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Default

Just add up to 5ul, or so, of your RNA to 19.5 ul of the "Elute,Prime,Fragment Mix" and start at the "Incubate RFP" step. Remember that your RNA is already somewhat degraded though. So you may want to cut back your fragmentation time. 4 minutes gave us fragment lengths around what I would have expected for an 8 minute fragmentation.

The other caveat is that by doing the polyA step, Illumina's kit is also doing a purification that would get rid of pretty much everything other than polyA RNA -- salts, organics, proteins. You do not get that benefit this way.

Nevertheless, if your RNA is reasonably pure, it probably won't be a big issue.

--
Phillip
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