[Simone78]

Hey Simone,

I recently saw a paper, where they put at 3-carbon spacer on the 3' end of the TSO for template switching RT. Supposedly it's to reduce "primer founded amplification." I have never seen this before. Do you have any opinion on that?

Hi,
as said above, the reference would be appreciated.
I never saw this paper you are talking about and therefore I can´t comment on it. I can just tell you that we tried to block the 3´-end of the TSO in different ways but the cDNA yield after RT was always much lower and we eventually gave up.

[Simone78]

Hi Sunpenguin,
Can you please give me the sequence for your TSO (without LNA) for SMartseq2? Where do you order it from? I am doing single cell RNAseq for immune cells and am having more primers dimers than cDNA for single cell even when I have biotinlylated all the primers. For bulk immune cells, 10 and above, it is fine.
Thanks!
Serena

I had the same problem when working with innate lymphoid cells. Immune cells are small and have much less RNA than, for example, cell lines.
My advice is to block all the oligos with biotin (all!), increasing the number of PCR cycles (23 should be fine), make your own magnetic beads (according to the Rohland & Reich, Genome Res 2010 paper) and using a buffer with a lower % of PEG in order to increase the cutoff (if you are interested I can give you the details), using an oligodT with "V" and not "VN" in the end to avoid weird pairing with the 2 rG in the end of the TSO, as suggested in some papers (for example, look for the "CATS" paper in another thread on this forum). I would also play with oligodT conc (reducing it sometimes help a lot) but I wouldn´t touch the TSO conc.
Some (but not all) of these changes are described in the method part of our last paper --> PMID: 26878113

/Simone

[skwek1]

I had the same problem when working with innate lymphoid cells. Immune cells are small and have much less RNA than, for example, cell lines.
My advice is to block all the oligos with biotin (all!), increasing the number of PCR cycles (23 should be fine), make your own magnetic beads (according to the Rohland & Reich, Genome Res 2010 paper) and using a buffer with a lower % of PEG in order to increase the cutoff (if you are interested I can give you the details), using an oligodT with "V" and not "VN" in the end to avoid weird pairing with the 2 rG in the end of the TSO, as suggested in some papers (for example, look for the "CATS" paper in another thread on this forum). I would also play with oligodT conc (reducing it sometimes help a lot) but I wouldn´t touch the TSO conc.
Some (but not all) of these changes are described in the method part of our last paper --> PMID: 26878113

/Simone

Hi Simone,
Thank you! I will try out your suggestions. Should I try TSO with no LNA?
Thanks,
Serena

[Simone78]

Hi Simone,
Thank you! I will try out your suggestions. Should I try TSO with no LNA?
Thanks,
Serena

that´s a good question! LNA-TSO definitely increases sensitivity, although it comes with some problems (strand invasion). Personally, I am still using the LNA-TSO but many people are of a different opinion (also here in this thread).

[anamar]

Hello to everybody. I've come many times to this thread and I thank everybody for sharing their knowledge.

I prepared Nextera XT libraries from a pool of single cells. I amplify with SMARTER v4, and in some cases had molecules > 1.5kb disturbing my bioanalyzer, migrating to the next well and making it hard to quantify cDNA.

Many of my libraries are OK. But some libraries have come up with a peak around 150bp and I am unsure whether this is "bird nesting", over/undertagmentation or any other artefact that may influence my output. I kindly ask for your feedback. I attached some bioanalyzer profiles. Thank you all in advance.

[SunPenguin]

Hey Simone,

DOI: 10.1038/ncomms11112

Is strand invasion a big issue in your hands? The paper that was cited before tested using 3 LNA contigs (+G+G+G), and I wonder if using the rGrG+G configuration more or less avoids that problem.

I am also looking around at these different template switching protocol. How important is it to clean the first strand cDNA after synthesis? Some protocols even try to chew away the TSOs with exonuclease (by including some rU's throughout the sequence).

For the bulk sample stuff, I've never cleaned the first strand cDNA, and everything seems to be okay. Is it something that's more important for single cells?

[SunPenguin]

Hey Serena,

I've been playing around with several different versions of the TSOs, but for the single cell stuff, I've been using basically the exact same sequence as published in the original Smart-seq2 paper, but with the 5'biosg addition. the blocked oligos more or less avoids the concatemers (though sometimes I still get a little bit).

I have been using poly(dT) with 3'VN though. Perhaps that's part of the issue.

From my experience though, if you're not getting results from single cell sort, but is consistently getting results from 10 cells or more, the more likely issue is the sorting. I'm sure you're already doing this, but you have to fastidiously calibrate your sorter for single cell sort (I personally use a tiny hole puncher to punch out small circles of PH papers to detect where exactly a droplet falls in the well. Extremely time consuming, but it's the only way I can ensure good sorting).

Also, snap freeze your samples after sort, if you don't already. At least, put it directly on a layer of dry ice asap after sorting.

Also switch to Maxima H- RT if you haven't already. We get better success rate (within samples of the same sorting session) with maxima RT than with SSII or with SmartScribe. 42 degrees is better than 50, for Maxima.

[Simone78]

Is strand invasion a big issue in your hands? The paper that was cited before tested using 3 LNA contigs (+G+G+G), and I wonder if using the rGrG+G configuration more or less avoids that problem.

I think the strand invasion is difficult to quantify but it is definitely a problem, even when using "only" one LNA-G.

I am also looking around at these different template switching protocol. How important is it to clean the first strand cDNA after synthesis? Some protocols even try to chew away the TSOs with exonuclease (by including some rU's throughout the sequence).

For the bulk sample stuff, I've never cleaned the first strand cDNA, and everything seems to be okay. Is it something that's more important for single cells?

I think doing the purification after 1st strand synthesis is not necessary, even in single cells. However, we observed sub-optimal performances of the KAPA Pol when trying to reduce the volume of the pre-ampl reaction too much. The reason is probably that the salt and additives from the RT are disturbing the PCR. If you dilute them by adding 1.5-2 volumes of PCR mix the reaction works very well. Increasing the final volume even more probably make things even better but it becomes very expensive when you need to process hundreds cells and we don´t do it.

Exonuclease I will digest only ssDNA in direction 3´-->5´, basically only the free oligos not bound to the cDNA. The Quarz-seq protocol uses ExoI after RT but they don´t do the strand switching so they have only the hybrid mRNA:cDNA at the end of the RT (and blunt ends). I tried to do it as well (only once) but the following PCR failed completely and I never repeated it again. I must have done something wrong because, in theory, the TSO is annealed to the cDNA and gets extended, so the 5´portion of the hybrid cDNA:mRNA should actually be dsDNA and therefore not accessible for the ExoI.

[skwek1]

Hey Serena,

I've been playing around with several different versions of the TSOs, but for the single cell stuff, I've been using basically the exact same sequence as published in the original Smart-seq2 paper, but with the 5'biosg addition. the blocked oligos more or less avoids the concatemers (though sometimes I still get a little bit).

I have been using poly(dT) with 3'VN though. Perhaps that's part of the issue.

From my experience though, if you're not getting results from single cell sort, but is consistently getting results from 10 cells or more, the more likely issue is the sorting. I'm sure you're already doing this, but you have to fastidiously calibrate your sorter for single cell sort (I personally use a tiny hole puncher to punch out small circles of PH papers to detect where exactly a droplet falls in the well. Extremely time consuming, but it's the only way I can ensure good sorting).

Also, snap freeze your samples after sort, if you don't already. At least, put it directly on a layer of dry ice asap after sorting.

Also switch to Maxima H- RT if you haven't already. We get better success rate (within samples of the same sorting session) with maxima RT than with SSII or with SmartScribe. 42 degrees is better than 50, for Maxima.

Hi SunPenguin,
Thank you for your response! I have been using oligodtVN, but Simone has suggested using just oligodtV without the N according to the CAT publication. What are your thoughts about this?

I calibrate my single cell sort with 100 cells onto a film on top of the plate. I can see it is not as good as you what you have suggested. Nevertheless there are application of cDNA from single cell with 21 cycles, just not a lot. After sorting, I vortex on a plate shaker, spin down and snap freeze on dry ice.

There are definitely less dimers now (but still some) when I biotinylated all the oligos. I'm not sure if concatamers are a problem for me as I see some jagged peaks and they are not constant like for concatamers.

Is this amount of cDNA enough to proceed for single cell? Please see attached bioanalyzer profile for 0 and 1 cell.

I will try 23 cycles as Simone as suggested, as well as rGrGrG without LNA, and also Maxima H RT as you have suggested.

Thank you!
Serena

[SunPenguin]

How much lysis/RT buffer do you sort into?

The poly(dT)VN vs without VN is also new to me. I can see how it can make some weird binding situation with the template switch oligo.

For what it's worth, Some people at the Broad think they both work the same.

[achamess]

I think the strand invasion is difficult to quantify but it is definitely a problem, even when using "only" one LNA-G.



I think doing the purification after 1st strand synthesis is not necessary, even in single cells. However, we observed sub-optimal performances of the KAPA Pol when trying to reduce the volume of the pre-ampl reaction too much. The reason is probably that the salt and additives from the RT are disturbing the PCR. If you dilute them by adding 1.5-2 volumes of PCR mix the reaction works very well. Increasing the final volume even more probably make things even better but it becomes very expensive when you need to process hundreds cells and we don´t do it.

Exonuclease I will digest only ssDNA in direction 3´-->5´, basically only the free oligos not bound to the cDNA. The Quarz-seq protocol uses ExoI after RT but they don´t do the strand switching so they have only the hybrid mRNA:cDNA at the end of the RT (and blunt ends). I tried to do it as well (only once) but the following PCR failed completely and I never repeated it again. I must have done something wrong because, in theory, the TSO is annealed to the cDNA and gets extended, so the 5´portion of the hybrid cDNA:mRNA should actually be dsDNA and therefore not accessible for the ExoI.

Hi. Thanks so much for all of the info you provide and for developing Smart-seq2.

I used Smart-seq2 the other day for amplifying mRNA from single neuronal nuclei, as well as bulk (100 nuclei). It worked great, the first time! The singles amplified very nicely. I used 21 cycles, as advised by Krishnaswami et al (2016) in their Nat. Protocol. I got plenty of cDNA, and nothing from the buffer that contained the nuclei (NTC).

I was really pleased. FYI, I'm using biotinylated TSO, ISPCR and oligoDT.

My question is about the maximum number of cells/nuclei I can use. From the Nat protocol, I see the recommendation to not dilute the lysis buffer any more than 0.5 ul. When I sorted, I was certainly below this. but if I wanted to increase the number of nuclei that I process in bulk (say, 1000+), I would be going up to about 5 ul of sorting buffer (PBS), which would mess up the concentrations for the downstream Smart-seq2 procedure.

So I was wondering if you have any guidance. Can I scale up? Or, alternatively, I could sort into more lysis buffer (say 5 ul lysis buffer), lyse the cells and homogenize their mRNA, and then take a fraction of that lysis solution (2 ul) for downstream prep. That way I would be getting a representation of more nuclei (say 1000) but with only a fraction of their input. Is that advisable?

Thanks so much for your help.

[skwek1]

How much lysis/RT buffer do you sort into?

The poly(dT)VN vs without VN is also new to me. I can see how it can make some weird binding situation with the template switch oligo.

For what it's worth, Some people at the Broad think they both work the same.

2ul as in the Smartseq2 protocol.

Thanks,
Serena

[SunPenguin]

And do you recover all of 2uL? I usually sort into 5 uL (better chance of hitting the buffer). By the time I freeze, thaw, and actually pipette in the volumes to do RT, I get about 2-3uL back (I pipette out the lysate from the plate, since I don't usually process a whole plate at a time, yet). Evaporation happens fast with such small volume, even during the wait time on the sorter. Or unless you're sorting into 384 well plates, then the format is a bit different.

[skwek1]

And do you recover all of 2uL? I usually sort into 5 uL (better chance of hitting the buffer). By the time I freeze, thaw, and actually pipette in the volumes to do RT, I get about 2-3uL back (I pipette out the lysate from the plate, since I don't usually process a whole plate at a time, yet). Evaporation happens fast with such small volume, even during the wait time on the sorter. Or unless you're sorting into 384 well plates, then the format is a bit different.

Hi SunPenguin,
I sort into 96 well PCR plates with 2 ul of lysis buffer containing TritonX and Rnase In (same % as in paper), froze down and thaw as needed. Upon thawing, I vortex on a shaker and spin down before removing the microamp clear adhesive film. I add 2ul of premixed oligodtVN and dNTP, and do all subsequent reactions in the same plate.
Thanks for working with me on this!
Serena

[naveenrajkumar]

[QUOTE=Simone78;191832]

I think doing the purification after 1st strand synthesis is not necessary, even in single cells. However, we observed sub-optimal performances of the KAPA Pol when trying to reduce the volume of the pre-ampl reaction too much. The reason is probably that the salt and additives from the RT are disturbing the PCR. If you dilute them by adding 1.5-2 volumes of PCR mix the reaction works very well. Increasing the final volume even more probably make things even better but it becomes very expensive when you need to process hundreds cells and we don´t do it.


Hi Simone,
what is the lowest total volume of preamp reaction have you successfully performed using KAPA? or when do you notice sub-optimal performance of KAPA. what was the ratio of RT in KAPA mix. I notice in your paper you use 20% cDNA in PCR volume (10ul in 50ul). Have you tried alternate enzymes to KAPA for LD PCR in low PCR volumes say 10 ul.

Thanks