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]

Originally Posted by 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.

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.

Originally Posted by 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.

[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

[liaojinyue]

We are buying reagents for smart-seq2. It turns out that TSO is not very cheap(~750usd for the smallest scale 250nmole)? Meanwhile I want to make sure there is nothing wrong with my technique otherwise will face the same problem in the future. Any idea what could possibly go wrong?

[liaojinyue]

Hi, Simone, we are testing SMART-seq2 protocol in our lab recently. It's quite impressive in terms of cDNA yield after preamplification. However, we notice that the our cDNA library is not as uniform as it should be. Although we can see it covers the range from several hundred to thousands, there are several intense peaks with the highest of around 1800bp.
We tried different TSO concentration and cycle numbers but did not see any improvement. Please see attachment 1 (cDNA is purified prior to bioanalyzer analysis)
We also tried to replace some component or add additional bead purification after first strand synthesis. The oligo dT30 primer provided in the clontech smart-seq kit did show some improvement while there are more concatmer formation with additional bead purification step. Please see testing result 2 (We want to see whether there is concatemer formation so we load the PCR product in bioanalyzer without purification).

I think I'm close to a successful run but also so desperate that I can not solve this problem. Could anyone please give me some suggestions? What are these bands and how to get rid of them? Thanks a lot.

Originally Posted by 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

[Simone78]

Hi,
what you saw here is nothing new for us and we still can´t explain what it is. we also saw a peak around 1.8 Kb when optimizing the protocol using HEK293T (human) cells. When we looked for over-represented sequences in the data we found out that the peak corresponded to a specific transcript called "humanin", which is mitochondrial (!) but has several homologous copies on the genome. While it´s annoying having such a library, the results were not affected. Below please find an example of what we got when we were optimizing Smart-seq2. There you can see we had not only this humanin peak but also a fair amount of primer dimers, but still the performance of our method was superior to the Clontech kit (primer dimers get tagmented with the Nextera kit as well and end up in the final library, thus "wasting" sequencing reads). Sometimes we see it also with mouse cells, but this obviously can´t be humanin which, as the name says, is present only in humans. For the mouse we never found out what it is...what we know is that it´s not a contamination because some of our collaborators reported the same. On the other hand, even when working with single cells this peaks doesn´t always come up. Maybe it depends on how stressed the cells are or how they were sorted? I would also be interested in finding it out!
In conclusion, Smart-seq2 is better than Smart-seq but it´s not perfect, sorry!

I saw that you tried to reduce the amount of TSO. I did few trials on the amount of the different primers. While reducing ISPCR and SMART dT30VN sometimes (sometimes!) helps in reducing the amount of primer dimers, decreasing the TSO usually leads to lower cDNA yield after preampl. Maybe the strand-switch reaction is inefficient and you need to have such a huge amount of TSO even when working with single cells!
Have you tried to sequence some of these libraries? I don´t know what you are interested in, but if you need a lot of reads you might simply multiplex less samples per lane. For most applications (diff expression, detection of isoforms/splice variants) 1 million reads/sample are sufficient and you would get that amount even when pooling 96 samples on a Illumina HiSeq 2000.

We had some concatamer problem in the beginning, but they were due to too much adaptors (TSO, ISCR or oligo dT) compared to the RNA of a cell and we observed that only with very small cells (mostly immune cells which have very little mRNA). If it is an issue, you might try to block the TSO at the 5´end as done by Kapteyn J et al (BMC Genomics 2010, 11:413). Blocking the 5´should prevent concatamerization of the TSO.

Best,
Simone

[jwfoley]

Maybe one possible problem is the LNAs in the TSO? This is prone to strand invasion (doi:10.1186/1471-2164-14-665), so the LNAs are not a good idea if you care about full-length transcripts. That's why all the other template-switching protocols use riboguanines. If the strand invasion is biased toward specific sites, that could explain these problems.

EDIT: on further inspection, those bands are definitely the characteristic "hedgehog" signal from using unblocked oligos. Block them and it should be fine. I don't know why there are any protocols floating around out there that say blocking is optional; it's so cheap to fix.

[Simone78]

Originally Posted by jwfoley

Maybe one possible problem is the LNAs in the TSO? This is prone to strand invasion (doi:10.1186/1471-2164-14-665), so the LNAs are not a good idea if you care about full-length transcripts. That's why all the other template-switching protocols use riboguanines. If the strand invasion is biased toward specific sites, that could explain these problems.

EDIT: on further inspection, those bands are definitely the characteristic "hedgehog" signal from using unblocked oligos. Block them and it should be fine. I don't know why there are any protocols floating around out there that say blocking is optional; it's so cheap to fix.

We use a LNA-containing TSO and always have a better coverage at the 5´-end of transcripts than the Clontech kit which uses 3 riboG.

In our hands, blocking the TSO led to a (slightly) lower gene detection at all RPKM levels and a (slightly) higher variation between replicates. The unblocked LNA-containing TSO was still performing better, therefore we don´t block the TSO unless the "hedgehog" signal shows up.