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Old 12-20-2011, 06:47 AM   #1
Claire1
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Location: Spain

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Default directional mRNA-Seq Sample Preparation

Hello,

I want to start to work on directional mRNA-Seq Sample Preparation.
I would like to know if anybody has experience with the Illumina protocol -directional mRNA-Seq Sample Preparation- and/or the protocol from epicentre -ScriptSeq™ mRNA-Seq Library Preparation Kit-.
In your experience which one gives you the best result.
Any suggestions are very welcome.
thanks!
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Old 01-25-2012, 01:18 PM   #2
Garyron
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Hi Claire1,
Have you decided on the kit , I just saw your post and illumina's directional protocol needs items part # from two kits as well as a fragmentation buffer but it works very well.
Bests
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Old 01-25-2012, 02:12 PM   #3
epistatic
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The Illumina protocol using multiple kits is expensive and I found the Epicentre protocol to be annoying for small sample numbers and not scalable for larger sample numbers. The Epicentre method has one long t-cycling program with wait times, pauses, + 1 ul to every tube multiple times. It is quick though (I could do libraries in one day) but I found painful.

I now use the Illumina mRNA TruSeq kit with modifications and it works very well.

I take the sample through the TruSeq kit per protocol through 1st strand. After 1st strand I clean up the sample with Zymo's RNA C&C -5 to remove dNTPs. I then do 2nd strand with a dNTP mix with dUTP (U, C, G, A) and NEB's dNTP-free 2nd Strand reaction buffer and 2nd Strand RNA-seq module. The resultant cDNA is taken back into the TruSeq kit until right before PCR. At this point you can treat the sample with USER and then go into PCR, or go directly into PCR (The kit taq looks to choke on dUTP strand, USER step likely not needed). This has been used on 100+ samples in my lab and works well.
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Old 02-02-2012, 01:47 AM   #4
Claire1
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Hello,
thanks for your replies!

I decided for the kit from epicentre. Before the Sample Prep I was doing a rRNA removal with the Ribo-Zero rRNA Removal Kit. Both protocols were easy to follow, fast and were working fine. Indeed the steps to pipete 1ul individually is not setted up for larger sample numbers, but this will be not a problem for us. Now we are waiting for the sequencing results.

Very interesting your modified mRNA TruSeq protocol. With USER do you mean UNG treatment ?

Thanks!
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