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Old 03-20-2017, 09:32 AM   #1
PrimerBuffalo
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Default Smart-seq2 sample prep questions

Hi All,

I have a couple questions about the Smart-seq2 method for amplifying single cells (Sandberg 2014), and I hope someone with experience in this method might have some answers:

1) Why are dNTPs present in the catching buffer that you sort cells into? Couldn't they just be added with the RTase after the initial binding of the OligoDT to the polyA tail?

2) What are the drawbacks of having OligoDTs or dNTPs in excess to what is written in the original protocol? I am worried about the concentrations not being enough for a highly transcriptional cell.


Thanks in advance for the help!
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Old 03-20-2017, 11:26 AM   #2
cmbetts
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1) If I remember right, they mention in the paper that they got a statistically significant yield increase adding dNTPs at that step. It definitely would still work, just with somewhat lower efficiency if it's added w/ RT
2) Too much dT can generate artifacts, more dNTPs probably wouldn't have a noticeable effect. That same protocol can be used without modifying the RT conditions for bulk samples with >10ng RNA (only PCR cycles are reduced), so you don't need to worry about insufficient primer for any single cell applications. There are several orders of magnitude more primer than mRNA in the cell. You should do some preliminary characterization experiments with your cells to dial in the correct number of PCR cycles, so you might end up shaving off a cycle or two from the default if your cells are particularly active.
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