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Old 11-19-2018, 08:08 AM   #4
Simone78
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Location: Basel (Switzerland)

Join Date: Oct 2010
Posts: 207
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Quote:
Originally Posted by Kaizen View Post
Do you have any experience using Smartseq2 for bulk samples? from 1000-10000 immune cells.
I would like to know what's the maximum number of cells that you can process in a 96 well plate using bead purification without modifiying the RT-PCR and also the maximum number of cycles recommended given a certain number of cells
Thanks
Hi,
Smart-seq2 works for bulk samples, however 10K cells might be too much. Among the possible drawbacks:
- lysis might be incomplete. To avoid this I would increase the % of Triton in the LB. You can surely increase it to 1-2% (as in the STRT-seq 2i protocol). Higher % of Triton seem also not to harm the RT enzyme but I would be cautious with that. Try to decrease the number of cells instead.
- You might run out of some components later in the pre-ampl reaction. The most likely "candidates" are dNTPs and oligos (all). I would suggest to run a sort of titration experiment, something along the line: if you start from 1K cells do you get more cDNA after pre-ampl when using 2 uM final of TSO vs 1 uM? I know itīs just empirical but so far I didnīt come up with something better, I am afraid. If you donīt want to modify the original protocol I would try to stay in the range 100-500 cells. Maybe it works also with 1K cells, but I would at least increase the % of Triton. While the amount of RNA/cell is known for many cell types (and reported to be around 10 pg tot RNA for cell lines), I couldnīt find reliable numbers for immune cells. probably 0.1-1 pg/cell?

With single T/B cells, ILC or monocytes I do 22-24 PCR cycles for the pre-ampl reaction. With >1 cell I would adjust the number of cycles accordingly. As a guideline: for 100 cells you might find 18-20 cycles sufficient; for 1K cells maybe 16-18, and so on. In the end you just want to have enough cDNA to be able to see it on the Agilent BA or TS. For tagmentation, both with the kit or the home-made Tn5 you would anyway use 1 ng cDNA or less. I would elute in smaller volumes after bead cleanup rather than increasing the number of pre-ampl cycles to be able to see it on the BA/TS.

I hope this helps!
/Simone
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