RNA-seq from single cells?

[irit]

Does anyone here have experience with this? Does anyone know of a good protocol with consistent results?

[CTC]

I would like to do this as well but have not figured it out. Have you?

[balaji]

are you looking for something like this.. Cell article, probably I should look for some public data if there is any?

[TonyBrooks]

I recommend using the Clontech SMARTer PCR kit (if you can afford it).
We have some nice data from single Molt4 cells.
I can't comment on consistency of data as we've only done it once with 4 cells.

[CTC]

Thanks for the article. But without detailed protocol it is hard to replicate the experiment.
I have tried SMARTer PCR kit but it did not work in my hands. The expected yield for a single cell is 7 ng, the negative control yield is similar.

[daantje]

Hi Yes, would also be interested in hearing if there are tricks for the SMART kit. I tried and with a 10ng input of control RNA, I only get 500pg of amplified cDNA. Even though the size is correct and I am happy to get to this point, still my yield is much lower then suggested in the manual. I have not yet proceeded with library generation.

[solidestcloud]

I'm not sure if any of you have seen this?http://www.fluidigm.com/home/fluidig...-6199%20A1.pdf
We have just released a Single Cell mRNAseq protocol for the C1 system. It utilises the SMARTer chemistry but miniaturises it making it much cheaper.

[MMaria]

I'm not sure if any of you have seen this?http://www.fluidigm.com/home/fluidig...-6199%20A1.pdf
We have just released a Single Cell mRNAseq protocol for the C1 system. It utilises the SMARTer chemistry but miniaturises it making it much cheaper.

Interesting. Does anybody knows how much does it cost or which labs already have it?

[kwaraska]

We are getting one in March but we are in Boston so that may not help you but it could help others.

[daantje]

Kwaraska - did you test the instrument? or have you seen generated data?

[kwaraska]

I have not tested it but we hosted a seminar with actual data and my boss has had meetings with the supplier and he has seen it in action.

[GW_OK]

Saw a demo of it at AGBT. $150k for the box is what I was told, with $23/sample all-in library costs. cDNA is generated by the SMARTer kit with the Illumina library made with Nextera from the cDNA. Does 96 cells in one go.

[Simone78]

a detailed protocol for single-cell seq is described in a paper published last year by our group: http://www.ncbi.nlm.nih.gov/pubmed/22820318
the CEL-Seq protocol (published in Cell Reports at the end of last year) is targeting only the 3´end of mRNAs, while the Smart-Seq aims to get full-length mRNAs. Another method, STRT (Islam et al., Genome Res. 2011 & Nat. Prot. 2012) captures the 5´-end only instead. It depends what one is looking for.

[liaojinyue]

Hi, daantje, have you figured out the cause of low yield? I tried the kit recently and also encountered similar problem, only got less than 1ng amplified cDNA from 500pg input RNA provided in the kit after 14 cycles. I would really appreciate if anyone can offer some suggestions for this kit.

Hi Yes, would also be interested in hearing if there are tricks for the SMART kit. I tried and with a 10ng input of control RNA, I only get 500pg of amplified cDNA. Even though the size is correct and I am happy to get to this point, still my yield is much lower then suggested in the manual. I have not yet proceeded with library generation.

[Simone78]

we have recently published a paper where we describe Smart-seq2, an improvement of the Smarter kit. Check it out! You don´need any kit to make your cDNA and you´re going to save a lot of money!

http://www.ncbi.nlm.nih.gov/pubmed/24385147

http://www.ncbi.nlm.nih.gov/pubmed/24056875