Does anyone here have experience with this? Does anyone know of a good protocol with consistent results?
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are you looking for something like this.. Cell article, probably I should look for some public data if there is any?
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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.
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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.
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Originally posted by solidestcloud View PostI'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.
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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.
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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 View PostHi 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.
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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!
Emerging methods for the accurate quantification of gene expression in individual cells hold promise for revealing the extent, function and origins of cell-to-cell variability. Different high-throughput methods for single-cell RNA-seq have been introduced that vary in coverage, sensitivity and multi …
Single-cell gene expression analyses hold promise for characterizing cellular heterogeneity, but current methods compromise on either the coverage, the sensitivity or the throughput. Here, we introduce Smart-seq2 with improved reverse transcription, template switching and preamplification to increas …
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