[weigrc]

Has Anyone tried the downstream DNA sample preparation Kit after SMARTer kit. Since the output of SMARTer kit is about some 10 ng, I am looking for the DNA prep kit for the downstream work. The one SMARTer kit recommonded need to have 1 ug sample requirement. Any more suitable DNA prep kit for the purpose??

[calcijw]

There are couple publications on the SMARTer Ultra Low RNA kit so far. The NBT paper also covered data and protocols using Nextera instead of PE protocol following the cDNA synthesis.

http://www.frontiersin.org/Neurogenomics/10.3389/fgene.2012.00124/abstract

http://www.nature.com/nbt/index.html

[canted]

There are couple publications on the SMARTer Ultra Low RNA kit so far. The NBT paper also covered data and protocols using Nextera instead of PE protocol following the cDNA synthesis.

http://www.frontiersin.org/Neurogenomics/10.3389/fgene.2012.00124/abstract

http://www.nature.com/nbt/index.html

Thanks for the response; I will check these papers.

[monikak]

check this...SMARTer technology

http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.2282.html

[anamar]

Hello I'm currently designing my experiment. I'm really a newbie so I apologise in advance for any nonsense. This threat has been really useful thanks.

I will check the papers but for a quick answer: does anyone know if you can barcode your libraries with the smarter kits, or in the library prep stage? which library prep kit will be suitable to use? in the illumina page it is suggested the Paired-End DNA Sample Prep Kit but I believe that barcoding of libraries can only be done with the TruSeq RNA Sample Prep Kit. Am I right?

Which strategies would you suggest? Thanks.

[HoneyBee]

You can use Multiplexing Sample Preparation Oligonucleotide Kit from Illumina for indexing your samples.

[weigrc]

The indexes can be added in the library preparation steps, not in SMARTer kit process.

And the PE DNA prep kit doesn't include indexed adaptors, but "pure" adaptors, the indexed ones need to be extra purchased... However, TruSeq DNA/RNA Kits includes indexed adaptors...

Firstly, by using SMARTer kit, you need to concern about the DNA output after SMARTer kit and find the compatiable library prep kit...

[bpinder]

Low Input RNA-seq

Hello all,

Newbie here trying to move forward with an RNA-seq project. Thank you for your patience.

I have samples containing ~50ng of total RNA for RNA-seq. I am considering the SMARTER and OVATION technologies and have several questions regarding the workflow:

1) When (and how) should I remove rRNA?
2) Do both kits support downstream barcoding for multiplexing?
3) Would it be possible to instead use Whole Genome Amplification followed by KAPA kit?

I very much appreciate your feedback.

Thank you,
Ben

[ajthomas]

T7 (Eberwine) RNA amplification is commonly used to amplify mRNA for analysis by microarrays, but I have not seen anyone use it to amplify RNA for RNA-Seq analysis. Is there any reason it wouldn't work?

[NextGenSeq]

The Eberwine method gives 3' biased libraries.

We routinely make RNA-Seq libraries from 1 ng of total RNA using the NuGen V2 RNA-Seq kits.