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Old 03-06-2014, 10:33 AM   #1
Agun
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Default Library prep kit for low input RNA seq

Hello.

We are aiming to perform RNA sequencing on stem cells. This means that it will be hard to get enough of starting material, somewhere between 50-100ng total RNA per replicate (Running for biological triplicates) is realistic to assume.

We would like to investigate differently expressed genes and non coding RNAs, this means that rRNA depletion is of interest on a HiSeq

I have been in contact with Illumina and they tell me that they guarantee that their Truseq works perfectly using 100ng input material but many of out samples will be around 50ng.

My question is which kit to use for this kind of "low but not ultra low" input material?


Thank you for helping.

Kind regards,

Anders

Last edited by Agun; 03-06-2014 at 10:35 AM.
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Old 03-07-2014, 03:04 AM   #2
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Anders, consider Epicentre's ScriptSeq kit.

http://www.epibio.com/applications/r...reparation-kit
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Old 03-07-2014, 08:13 AM   #3
Jean
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We've used the Epicentre ScriptSeq2 kit for 50ng of mRNA with good results
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Old 03-07-2014, 11:29 AM   #4
Genohub
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It would be nice to see a data comparison of the "ultra-low" vs "low" input RNA-Seq kits. Any experience ? It certainly sounds like there is some concern that "ultra-low" isn't appropriate for all low input samples.

- Genohub
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Old 03-10-2014, 07:02 AM   #5
Agun
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Is there any reason to struggle to gather 100ng (Truseq) if Epicentre also uses rRNA depleted with a lot lower starting amount?

What is your experience with regard to normal data? We might otherwise do single cell profiling.


Thank you for your answer.
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Old 03-10-2014, 08:26 AM   #6
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As long as your rRNA depletion method works well, I would use the ScriptSeq method. The kit, in our hands, performs well at these lower amounts.
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Old 03-12-2014, 11:33 AM   #7
Agun
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Thank you for all your answers.

I have spoken to some persons who told me that I should keep out from making cDNA library my self since it can take several months to make it work.

So I therefore have to ask again with some additions:
- I do not have _any experience with library preparation or anything close to it_ nor do I have anybody here that knows how to do it and it is not possible for me to put several months for optimizing library preparation.
- I have standard laboratory experience for a started PhD.
- I have 50ng RNA starting material per sample
- I want to investigate differently expressed genes, splice variants and if possible non coding RNAs (the non coding RNAs one goes away quickly if a polyA-isolation kit is the easiest one)

Based on these very important facts, do you still advice me to try the kits you recommended?

Lastly and also important: If I make my own libarary, in which steps can I see if it worked well? Do I need to perform the whole sequencing and validate the genes with qPCR before one can say anything or is it possible just by doing a QC before sequencing?

Thank you very much for your help!

Kind regards,

Anders

Last edited by Agun; 03-12-2014 at 11:55 AM.
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Old 03-12-2014, 01:14 PM   #8
Genohub
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Hi Anders,

It depends on how precious your RNA sample is. If it's not that precious and you can get more of it, I would say use the kit recommended in this thread and go for it. Always good to start somewhere and library prep will be good molecular biology experience for someone starting a PhD program (just read about what each enzyme does at every step so it's a good learning experience and not just following a kit cook book). If your sample is really precious I would recommend letting an experienced service provider prepare your library. If you need recommendations fill out our consultation form and we'll connect you to someone who has experience with low input RNA library prep: https://genohub.com/ngs-consultation/

- Genohub
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Old 03-13-2014, 10:53 AM   #9
Agun
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We can get more of the sample and it would be nice to try to make my own library if that is possible without having a core facility as support.

The very important question is still: how do I know if everything went right, do I have to sequence every sample first or can a bioanalyzer and QC report pre-sequencing show me if I generated a good library?

Thanks for answer
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Old 03-13-2014, 12:26 PM   #10
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After you make the library, I would run it on a bioanalyzer to look at your library size distribution and then do qPCR to calculate an exact concentration prior to sequencing. The core facility you're working with may be willing to do the qPCR for you (or might do it regardless of if you performed one yourself).

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