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Old 03-04-2020, 01:50 AM   #281
Luke017
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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 01:53 AM.
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Old 03-04-2020, 02:47 AM   #282
Simone78
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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 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.
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, 03:44 AM   #283
Luke017
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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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Old 08-24-2020, 01:30 AM   #284
Luke017
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Default PhiX Spike Recommendations

Hi All,

I just wanted to say thank you for the help, with all of the tips in this thread I have now managed to generate good qualit cDNA and what should hopefully be some nice libraries!

I was wondering if anyone could help me out with one final question though, I have been working solely with memory B cells, and I have sent the libraries to our genomics facility. The genomics facility got in touch to say that they use 1% PhiX spike in for their sequencing runs but that low diversity libraries normally require a bit more. I think my final library is going to be pretty low diversity so I was wondering if anyone had any recommendations on what PhiX % to try?

Any help would again be much appreciated!

Best wishes,

Luke
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Old 08-24-2020, 10:42 AM   #285
cmbetts
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The random tagmentation (or sonication/ligation) will give it plenty of diversity. Lower diversity library types needing extra PhiX are things like amplicons, microbiome 16S, and WGBS.
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Old 08-24-2020, 10:54 AM   #286
Luke017
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Default Thanks!

Ah, that makes sense, thanks! I am clearly still thinking on too big a scale, I’m sure a few more months working on the sequencing aspect and I’ll get used to it.
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Old 01-19-2021, 07:29 AM   #287
Luke017
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Default Tagmentation/Index PCR Volume Cutting

Hi All,

I hope you are all doing OK in these challenging times and managing to keep busy!

With the help of this thread I managed to carry out my first successful Smart Seq 2 run. I am now just going through my protocol and adding up costs etc and I noticed in the updated 2019 protocol the tagmentation and index PCR step uses 10 times less of the nextera kit reagents whilst the amount of cDNA recommended (50-150pg is pretty much the same) compared to original 2012 publication. I obviously trust this recommendation but I just wanted to check if anyone has experience with this? This would basically make the kit last 10x longer than the supplier recommendation which just seems too good to be true! Also sadly I would need to hand pipette these small volumes so not sure if this cut down recommendation is only for liquid handling robot platforms?

Any offers of advice/help would be once again greatly appreciated!

Luke
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Old 01-19-2021, 10:30 AM   #288
Simone78
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Quote:
Originally Posted by Luke017 View Post
Hi All,

I hope you are all doing OK in these challenging times and managing to keep busy!

With the help of this thread I managed to carry out my first successful Smart Seq 2 run. I am now just going through my protocol and adding up costs etc and I noticed in the updated 2019 protocol the tagmentation and index PCR step uses 10 times less of the nextera kit reagents whilst the amount of cDNA recommended (50-150pg is pretty much the same) compared to original 2012 publication. I obviously trust this recommendation but I just wanted to check if anyone has experience with this? This would basically make the kit last 10x longer than the supplier recommendation which just seems too good to be true! Also sadly I would need to hand pipette these small volumes so not sure if this cut down recommendation is only for liquid handling robot platforms?

Any offers of advice/help would be once again greatly appreciated!

Luke
Hi Luke,
thatīs correct, itīs not a mistake. When starting from 50-150 pg cDNA you donīt need to use the standard amount of i5/i7. We generally dilute the adaptors 1:5 with water or low-EDTA TE buffer. We then make "working dilution" plates with already unique i5/i7 combinations in each well. You can then pipette them with a multichannel but of course it would be easier with a robot.

However, the Illumina i5/i7 allows you only to pool 384 cells at the most (24 i7 x 16 i5). We now generated additional sets of adaptors and ordered from IDT. Then we do something similar as described above, with the exception that we now know the initial concentration (the 1:5 dilution is the result of "trial and error", there is nothing scientific behind this particular ratio).
We dilute all the adaptors to 10 uM and combine equal volumes of i5 and i7 in working dilution plates. Again, each well has a unique combination of i5/i7, each at 5 uM conc. We then use 1 ul of this mix for a 5 ul Nextera reaction (0.5 ul cDNA, 0.5 ul ATM, 1 ul TD, 0.5 ul NT, 1.5 ul NPM, 1 ul i5/i7). If you use the Nextera ones you would still use 1 ul of 1:5 diluted i5/i7.

I hope this helps!
Best,
Simone
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Old 01-21-2021, 05:47 AM   #289
Luke017
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Location: London

Join Date: Jan 2020
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Default

Quote:
Originally Posted by Simone78 View Post
Hi Luke,
thatīs correct, itīs not a mistake. When starting from 50-150 pg cDNA you donīt need to use the standard amount of i5/i7. We generally dilute the adaptors 1:5 with water or low-EDTA TE buffer. We then make "working dilution" plates with already unique i5/i7 combinations in each well. You can then pipette them with a multichannel but of course it would be easier with a robot.

However, the Illumina i5/i7 allows you only to pool 384 cells at the most (24 i7 x 16 i5). We now generated additional sets of adaptors and ordered from IDT. Then we do something similar as described above, with the exception that we now know the initial concentration (the 1:5 dilution is the result of "trial and error", there is nothing scientific behind this particular ratio).
We dilute all the adaptors to 10 uM and combine equal volumes of i5 and i7 in working dilution plates. Again, each well has a unique combination of i5/i7, each at 5 uM conc. We then use 1 ul of this mix for a 5 ul Nextera reaction (0.5 ul cDNA, 0.5 ul ATM, 1 ul TD, 0.5 ul NT, 1.5 ul NPM, 1 ul i5/i7). If you use the Nextera ones you would still use 1 ul of 1:5 diluted i5/i7.

I hope this helps!
Best,
Simone

Ah, this is amazing, thanks again for all of your help.
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Old 02-16-2021, 09:35 AM   #290
Luke017
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Default Adaptor sequencing issue

Hi all,

Apologies for my constant requirement for help (the downside of not having a core sequencing facility within my department). Following my first Smart Seq 2 test run which was largely successful the bioinformatician who is doing the analysis pointed out that a higher proportion than expected of my sequencing is adapter (Fast QC plot attached). I was carrying out 150bp paired end sequencing so obviously this resulted in quite a lot of wasted sequencing. I was wondering if anyone else has had a similar issue and suggestions on adaptions I could make to avoid this? I was rather surprised as my tapestation traces of my library following tagmentation and index addition PCR were largely of the expected size with few fragments <100 bp (traces attached).

Maybe this is a more general issue that I should post in a different thread but I thought I might first see if anyone had any advice from a Smart Seq 2 point of view.

Once again and hopefully for a final time any input/suggestions anyone has would be much appreciated.

Luke

fastqc_adapter_content_plot.jpg 2020-08-11 Luke Smart Seq 2 Opti plate 6 Post Tagment and Index PCR.pdf
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