Originally posted by Simone78
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However, if we speak about mRNA-seq from ultra-low (1-100) number of cells, then SMART-seq is probably the most convenient way (and Ribozero would not be even necessary). However, most researchers are working with much higher number of cells (e.g. growing on 96 - 24well plates), from where obtaining 100-1000 ng of RNA is not a problem. After ~30 min procedure of poly(A)-enrichment, one can get 3 – 30 ng of mRNA in 40 µl eluate, and run CATS even without a need to concentrate the polyadenylated product before RT. Also, unlike SMART-seq, CATS gives (1) strand-specific information about mRNA and (2) has even coverage along all mRNAs. While:
(1) SMART-based mRNA-seq is not strand specific.
(2) with SMART-seq 5’-proximal and 3’-proximal parts of mRNAs are likely to be significantly underrepresented due to the inevitable premature template switching and taqmentation bias. Please correct us if we are wrong.
(3) SMART-seq is limited only to mRNA-sequencing; while CATS allows any RNA-seq, including small (20-200nt) RNA, like RIP-samples, miRNA, piRNA etc, and also any DNA-seq. So it is a universal protocol.
(4) SMART-seq would actually require more efforts because “RT and template switch” there is used only to generate and pre-amplify long cDNAs from mRNA. The library preparation itself occurs afterwards via fragmentation/adaptors ligation or taqmentation and further pre-amplification + purification. It is much easier to do mRNA enrichment (30 min), fragmentation/cleanup (20 min) and CATS (4-5 hours total, 20 min hands-on time).
To summarize, if you have a few cells and require only mRNA-seq than SMART-seq is the probably the best option. However, one can still convert mRNA from a single-cell into cDNA using poly(dT) primers, and run CATS after genome-wide DNA pre-amplification till hundred picograms.
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