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Old 10-27-2010, 04:42 PM   #1
upendra_35
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Default ScriptSeq mRNA-Seq Library Preparation Kit

Hi

I am wondering has anyone tried using this kit to make RNA-Seq libraries? They say it is quick (~3hrs) and also strand specific.
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Old 10-28-2010, 03:44 PM   #2
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I will be using it in the coming weeks from samples we have other RNA data on already--so I should be able to give you a good response in say, a month?

Let me know if you find anyone else as well.
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Old 10-29-2010, 12:55 PM   #3
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It is very easy to use. I'm analyzing our first RNA-Seq libraries made using it now.
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Old 10-30-2010, 12:16 AM   #4
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Thanks both of you for the responses. How did you or plan to multiplex your samples for sequencing as it has totally different method of reading barcodes with an extra MIR primer. I have ordered the kit and came to know the Genome centre in our campus doesn't use MIR to sequence the library and i am totally confused of how to multiplex my samples for sequencing. Any information on this would be appreciated.
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Old 11-02-2010, 07:07 AM   #5
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Barcodes are added during PCR using distinct barcode PCR Primers to minimize representation bias of individual libraries. To sequence the library tags and the index/barcode sequences, you would use existing Illumina instrument reagents that include the Multiplex Read 1 Primer and the Multiplex Index Read Primer; for paired-end sequencing, use the Multiplex Read 2 Primer.

There is a good overview of multiplexing on Illumina's website. Also, if you still have questions, I can send you (via PM) the latest version of the protocol, which provides a little more detail on the primers.
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Old 11-02-2010, 10:01 AM   #6
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The initial RiboZero rRNA removal worked very well. The highest expressed gene is mitochondrial RNA followed by rRNA. However, these are within acceptable levels. I'm currently comparing the Epicentre RNA-Seq results to Illumina RNA-Seq and an Affy expression chip.
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Old 11-19-2010, 07:11 AM   #7
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The description of the kit sounds really attractive. Has someone got good results with it already? I am trying to establish an RNA seq protocol in the lab, would be so nice to hear a couple of good references...
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Old 12-10-2010, 02:02 PM   #8
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Default Help on ScripseqTM

Quote:
Originally Posted by spenserbsmith View Post
I will be using it in the coming weeks from samples we have other RNA data on already--so I should be able to give you a good response in say, a month?

Let me know if you find anyone else as well.


So I was wondering whether you have finished the trial and how was it...I am in the middle to decide whether i shall give it a try, thanks, anyone?
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Old 12-10-2010, 04:33 PM   #9
spenserbsmith
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I have run the scriptseq with both Poly A+ enrichment and the ribozero kits. So far, neither has worked at the level we hoped for; however, I'm am new at generating libararies for sequencing, so I'm not totally confident in my results. A close colleauge of mine has done a full RZ+SS run and got good libraries, but still with more reads of Ribosomes than epicentre got in their results.

I suggest you try a run yourself too--I'll continue to update others as we progress, if we continue to use this method. It doesn't take too long to perform this procedure (though it does have a significant cost, doesn't it?)
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Old 12-11-2010, 10:29 AM   #10
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Thanks a lot---when u were saying neither has worked at the level you hoped for...could you please elaborate a bit more on that point...also, u said your friend got good one, so what's the percentage of reads he got for rRNA even more than epicentre...

BTW, I have no hands-on experience with the tech at all although i am relative senior and pretty experienced with lots of other stuff...As well, we are stretching the budgets as well, there isn't much room for trial and error in our cases as I am trying to get out as much as possible to meet both my boss's interests and my own goal for my own independent career.


Quote:
Originally Posted by spenserbsmith View Post
I have run the scriptseq with both Poly A+ enrichment and the ribozero kits. So far, neither has worked at the level we hoped for; however, I'm am new at generating libararies for sequencing, so I'm not totally confident in my results. A close colleauge of mine has done a full RZ+SS run and got good libraries, but still with more reads of Ribosomes than epicentre got in their results.

I suggest you try a run yourself too--I'll continue to update others as we progress, if we continue to use this method. It doesn't take too long to perform this procedure (though it does have a significant cost, doesn't it?)
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Old 12-11-2010, 10:30 AM   #11
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Quote:
Originally Posted by NextGenSeq View Post
It is very easy to use. I'm analyzing our first RNA-Seq libraries made using it now.

So I was wondering whether you have finished the trial and how was it...I am in the middle to decide whether i shall give it a try, thanks,
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Old 12-11-2010, 10:33 AM   #12
gao331
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Quote:
Originally Posted by NextGenSeq View Post
The initial RiboZero rRNA removal worked very well. The highest expressed gene is mitochondrial RNA followed by rRNA. However, these are within acceptable levels. I'm currently comparing the Epicentre RNA-Seq results to Illumina RNA-Seq and an Affy expression chip.

Thanks again, any updates on your comparison among thee three?

As well, would you recommend the epicentre Seq library kit then?
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Old 12-13-2010, 07:02 AM   #13
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The advantage is that you see all the expressed transcripts. This is a disadvantage if your RNA has mycoplasma contamination which is very common in cultured cells.
Since there is no gel purification we prefer it over the Illumina kit.
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Old 12-14-2010, 05:40 PM   #14
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Hi NextGeSeq: Did you do any modifications for the SS protocol or just followed the manufacturer's instructions. If you did any modifications is it possible that i can share your modified protocol. We wanted to compare the SS method to illumina method
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Old 12-15-2010, 06:38 AM   #15
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We didn't modify the protocol we received. However, it was a beta kit so Epicentre may have made minor changes to the released protocol.
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Old 12-15-2010, 11:43 AM   #16
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Mycoplasma contamination will cause problems with any random-primed library prep method, due to the relative abundance of the mycoplasma RNA.

@gao331: If you are not sure about the ScriptSeq kit, we do have an evaluation program. Please PM me for details.
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Old 12-16-2010, 12:44 AM   #17
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Works well, reads are oriented off the negative strand and will appear as blue for positive and yellow for negative reads in GS.
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Old 12-21-2010, 01:20 PM   #18
upendra_35
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[QUOTE=spenserbsmith;31174]I have run the scriptseq with both Poly A+ enrichment and the ribozero kits. So far, neither has worked at the level we hoped for; however, I'm am new at generating libararies for sequencing, so I'm not totally confident in my results. A close colleauge of mine has done a full RZ+SS run and got good libraries, but still with more reads of Ribosomes than epicentre got in their results.

Hi i have recently tried to use Scriptseq for making RNAseq libraries but unfortunately none of my libraries worked with the kit. I am wondering if there are modifications you made for the Scriptseq kit? Any help would be appreciated.
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Old 12-21-2010, 01:21 PM   #19
upendra_35
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Hi i have recently tried to use Scriptseq for making RNAseq libraries but unfortunately none of my libraries worked with the kit. I am wondering if there are modifications you made for the Scriptseq kit? Any help would be appreciated.
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Old 12-22-2010, 08:05 AM   #20
epibio
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@Upendra_35: Please contact our technical services scientists at 1-800-284-8474 so we can discuss why you couldn't get the kit to work. There are many issues that affect the quality of a RNA-Seq library, so it would be easier to determine the problem if we had detailed information.
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