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Old 03-04-2020, 12:50 AM   #281
Luke017
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Location: London

Join Date: Jan 2020
Posts: 2
Default Smart Seq2 Lymphocyte Advice

Hi Simone & All,

I appreciate this thread has been quiet for a while but I hope someone out there is still occasionally reading it.

I have recently been attempting to carry out Smart Seq2 with single memory B cells. I can very efficiently clone out Ig genes via PCR following the pre-amplification suggesting I have at least some good quality cDNA but my traces appear dominated by primer dimer. I have been following the protocol exactly as published in 2012 Nature protocols and wish I had found this thread sooner as it has plenty of great tips especially for adapting the protocol to work with lymphocytes and avoiding the particular issue I am having.

In terms of obtaining good quality cDNA with minimal primer dimer following pre-amplification and clean-up it looks like the advice has changed over the years and having gone through every post I have decided to make the following adaptions for working with resting memory B cells which I suspect are going to have very little starting RNA to work with and I just wanted to check that I have understood all of these adaptions correctly?

1) Add 5’ Biotin to TSO, Oligo-dT30VN, and ISPCR primers

2) Titrate the number of pre-amp cycles, typically around 23-24 cycles for lymphocytes but will vary depending on the cell

3) Try titrating the Oligo-dT primer but do not alter the concentration of the TSO or ISPCR primers

4) Using an oligodT with "V" and not "VN" in the end

5) Make your own magnetic beads (according to the Rohland & Reich, Genome Res 2010 paper) and using a buffer with a lower % of PEG.
a. I was wondering if anyone knew how much of a difference this made? I’m sure it is more straightforward than it sounds but I like the ease of just going straight to the AMPure beads, but happy to make all alterations that will help my results


Any help anyone can offer would be much appreciated!

Best wishes,

Luke

P.S Thank you all for your wonderful input over the years to this thread, it has been so useful and filled a lot of gaps in our troubleshooting where we have sadly lacked replies from scientists in our local area working with this technique.

Last edited by Luke017; 03-04-2020 at 12:53 AM.
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Old 03-04-2020, 01:47 AM   #282
Simone78
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Location: Basel (Switzerland)

Join Date: Oct 2010
Posts: 206
Default

Quote:
Originally Posted by Luke017 View Post
Hi Simone & All,

I appreciate this thread has been quiet for a while but I hope someone out there is still occasionally reading it.

I have recently been attempting to carry out Smart Seq2 with single memory B cells. I can very efficiently clone out Ig genes via PCR following the pre-amplification suggesting I have at least some good quality cDNA but my traces appear dominated by primer dimer. I have been following the protocol exactly as published in 2012 Nature protocols and wish I had found this thread sooner as it has plenty of great tips especially for adapting the protocol to work with lymphocytes and avoiding the particular issue I am having.

In terms of obtaining good quality cDNA with minimal primer dimer following pre-amplification and clean-up it looks like the advice has changed over the years and having gone through every post I have decided to make the following adaptions for working with resting memory B cells which I suspect are going to have very little starting RNA to work with and I just wanted to check that I have understood all of these adaptions correctly?

1) Add 5í Biotin to TSO, Oligo-dT30VN, and ISPCR primers

2) Titrate the number of pre-amp cycles, typically around 23-24 cycles for lymphocytes but will vary depending on the cell

3) Try titrating the Oligo-dT primer but do not alter the concentration of the TSO or ISPCR primers

4) Using an oligodT with "V" and not "VN" in the end

5) Make your own magnetic beads (according to the Rohland & Reich, Genome Res 2010 paper) and using a buffer with a lower % of PEG.
a. I was wondering if anyone knew how much of a difference this made? Iím sure it is more straightforward than it sounds but I like the ease of just going straight to the AMPure beads, but happy to make all alterations that will help my results


Any help anyone can offer would be much appreciated!

Best wishes,

Luke

P.S Thank you all for your wonderful input over the years to this thread, it has been so useful and filled a lot of gaps in our troubleshooting where we have sadly lacked replies from scientists in our local area working with this technique.
Hi,
actually someone is still reading this thread
To answer your questions:
1- that's good!
2- 23-24 cycles is perfect, we never did more than that.
3- that might help as well. I would even increase the TSO conc and see if things get better, maybe from 1 uM to 2 uM.
4- debatable, don't agree with that but many people have a different opinion
5- lower PEG = lower recovery of small fragments, but also loss of longer fragments if the % is reduced too much. Something in the range 17-21% PEG 8K should be fine. You can also play the DNA:bead ratio, in case.

Please take a look at the updated Smart-seq2 in Meth.Mol.Biol., in case you haven't seen it: https://www.ncbi.nlm.nih.gov/pubmed/31028630

Good luck!
Simone
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Old 03-04-2020, 02:44 AM   #283
Luke017
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Location: London

Join Date: Jan 2020
Posts: 2
Default

Perfect, thank you so much for the advice, I really appreciate it and will put it all into action!

Best wishes,

Luke
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