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Old 07-16-2012, 09:31 PM   #1
Staroselec
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Location: Novosibirsk

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Default RNA shearing

Hello
I would like to fragment RNA samples into ~30-50 bp fragments prior to making cDNA libraries and sequencing with SOLiD.
Is it possible with the Covaris? What conditions? Could you advise other methods?
Thank you for your time and tolerance.
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Old 07-17-2012, 06:47 AM   #2
JPC
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You will have to get the whole transcription Library prep kit from ABI so I would stick with the enzyme shear that they recommend (using RNAseIII for 3 mins at 37degrees C).

The 3 min incubation should shear your samples between 25 and 750bp with the majority of the product towards the 100bp region. It will probably take a little trial and error but I would try adding 15 seconds to the incubation and see how far the peak shifts?
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Old 06-26-2014, 06:05 AM   #3
kandasamyalkondan
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Hello
I would like to fragment RNA samples into ~30-50 bp fragments prior to making cDNA libraries and sequencing with illumina.
Is it possible with the Q800R sonicator? .is there is any protocol
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Old 07-01-2014, 08:20 AM   #4
NextGenSeq
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I would make cDNA and shear it. Why do you want fragments that short?
You will lose them using Ampure bead purification as well as many column methods.
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Old 07-02-2014, 06:49 AM   #5
jwfoley
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Why don't you just heat your RNA in a magnesium solution? Or if you like kit science, Ambion makes a "fragmentation reagent" and that's pretty much what it is.

But these fragments will be too short for a regular sequencing library, so I suggest you revisit your experimental design, unless there's some really good reason why you need them to be so short.
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