[kobeho24]

Hi Gary,
I just tried with 0.2% SDS, in the same way as I am doing after tagmention to strip the Tn5 off the DNA before PCR and it didn´t work. I also tried SDS + NaOH/HCl (skipping columns or precipitation, as they recommend in the protocol) but, as I said, there was probably too much salt at that point and the PCR didn´t work.
/Simone

Hi Simone,
I don't know if you noticed that the newly published TGIRT paper in the journal RNA. They use the TGIRT to do the RT of human plasma RNA. And I realized that there is a fragmentation step prior to RT when dealing with whole cell total RNA as they suggested. Thus, on my perspective, defeat its better processivity compared to retroviral RT. You mentioned that the average size of cDNA generated by TGIRT is 4-5k. I just wonder if you have an in-house protocol to do so. It would be much appreciated if you let me know.
BTW, I noticed that the TGIRT can template switch not only RNA but also DNA, which means it might not be compatible with single-cell RNA-seq, unless we extract the total RNA from a single cell and do rRNA-depletion before RT, am I right?

Best!
Gary

[amcg]

Hi all,
I'm hoping to use the Smart-seq2 protocol to make rna-seq libraries from small populations (200-500) of cells collected by FACS. In this case would you advise sorting them directly into the lysis buffer (triton-x and RNase inhibitor) described in the Nature Protocols paper, or would it be better to extract the RNA first and then proceed with the reverse transcription on purified RNA?

Many thanks!

[chaichontat]

Hi Simone,

I used the Smart-seq2 protocol using the Superscript IV. For some reason, the reaction failed completely (no cDNA products were observed). When I did not add 1M betaine (Sigma) and 6mM MgCl2, the RT reaction went beautifully, but there's no template switching. The template was 10 ng of purified PBMCs RNA and the TSO was synthesized by Eurogentec. Could you provide me with the reaction conditions you used with SSRT IV?

Thanks!
Chaichontat

[Simone78]

Hi all,
I'm hoping to use the Smart-seq2 protocol to make rna-seq libraries from small populations (200-500) of cells collected by FACS. In this case would you advise sorting them directly into the lysis buffer (triton-x and RNase inhibitor) described in the Nature Protocols paper, or would it be better to extract the RNA first and then proceed with the reverse transcription on purified RNA?

Many thanks!

Hi,
even if your cells have a lot of RNA I wouldn´t do it. This is only my opinion, others might think differently. Every time you transfer RNA before any amplification step you lose molecules. I would sort directly into the lysis buffer, but be aware that 0.2% Triton might not be sufficient for lysing so many cells.
Best,
Simone

[Simone78]

Hi Simone,

I used the Smart-seq2 protocol using the Superscript IV. For some reason, the reaction failed completely (no cDNA products were observed). When I did not add 1M betaine (Sigma) and 6mM MgCl2, the RT reaction went beautifully, but there's no template switching. The template was 10 ng of purified PBMCs RNA and the TSO was synthesized by Eurogentec. Could you provide me with the reaction conditions you used with SSRT IV?

Thanks!
Chaichontat

Hi,
I did exactly as described in the Nat Prot paper, just replacing SSRTII with SSRTIV and doing the RT for 15 min @ 50 degrees and inactivated the enzyme for 10 min @ 80 degrees.. I tested it only on tot RNA (10 pg) and I actually noticed that when I didn´t add betaine and MgCl2 I got worse results than when using them...the opposite of what you found. in my case the template switching went ok, at least based on the cDNA yield and size.
Best,
Simone

[Simone78]

Hi Simone,
I don't know if you noticed that the newly published TGIRT paper in the journal RNA. They use the TGIRT to do the RT of human plasma RNA. And I realized that there is a fragmentation step prior to RT when dealing with whole cell total RNA as they suggested. Thus, on my perspective, defeat its better processivity compared to retroviral RT. You mentioned that the average size of cDNA generated by TGIRT is 4-5k. I just wonder if you have an in-house protocol to do so. It would be much appreciated if you let me know.
BTW, I noticed that the TGIRT can template switch not only RNA but also DNA, which means it might not be compatible with single-cell RNA-seq, unless we extract the total RNA from a single cell and do rRNA-depletion before RT, am I right?

Best!
Gary

Hi Gary,
yes, I saw the paper, thanks! I didn´t know that the TGIRT can do strand switch on DNA as well (this was not mentioned when I contacted the Lambowitz lab). When I first did my tests some time ago I used tot RNA. Unfortunately, it didn´t work at single cell level (pg) and I just dropped it. therefore, I never sequenced anything and never tried on a real cell. I do have a protocol that worked with 100 ng (!) RNA but after the publication of this new article, I want to try again using some modifications and see if I can get it to work with less RNA. So I can´t really share it right now, sorry!
Best,
Simone

[chaichontat]

Hi,
I did exactly as described in the Nat Prot paper, just replacing SSRTII with SSRTIV and doing the RT for 15 min @ 50 degrees and inactivated the enzyme for 10 min @ 80 degrees.. I tested it only on tot RNA (10 pg) and I actually noticed that when I didn´t add betaine and MgCl2 I got worse results than when using them...the opposite of what you found. in my case the template switching went ok, at least based on the cDNA yield and size.
Best,
Simone

Hi Simone,

It turned out that my PCR primers were the problem. I was too fixated on the template switching process that I completely ignored the preamplification. Upon replacing the primers, everything works beautifully. I really thank you for this brilliant protocol!

Chaichontat

[KroSeq]

SSRT IV vs SSRT II

Hi,
I did exactly as described in the Nat Prot paper, just replacing SSRTII with SSRTIV and doing the RT for 15 min @ 50 degrees and inactivated the enzyme for 10 min @ 80 degrees.. I tested it only on tot RNA (10 pg) and I actually noticed that when I didn´t add betaine and MgCl2 I got worse results than when using them...the opposite of what you found. in my case the template switching went ok, at least based on the cDNA yield and size.
Best,
Simone

Hej Simone,
what is the benefit of using SSRT IV instead of SSRT II?

[Simone78]

Hej Simone,
what is the benefit of using SSRT IV instead of SSRT II?

the RT reaction is way faster than with the SSRTII but, apart from that, I don´t see any improvement in the reaction (at least with my settings!). The prices are almost the same, the SSRTIV is a bit more expensive.

[longwood]

Superscript II contamination update

E. Coli contamination of superscript II has been confirmed by the manufacturer. I know a bunch of people reported this on this forum a few months back, so thought I'd share this letter (attached) I received from Thermo Fisher. The letter basically says they have started testing for E. Coli DNA as part of their routine test now and those who received the tainted lots will be sent free replacement upon request.

[kobeho24]

Hi Gary,
yes, I saw the paper, thanks! I didn´t know that the TGIRT can do strand switch on DNA as well (this was not mentioned when I contacted the Lambowitz lab). When I first did my tests some time ago I used tot RNA. Unfortunately, it didn´t work at single cell level (pg) and I just dropped it. therefore, I never sequenced anything and never tried on a real cell. I do have a protocol that worked with 100 ng (!) RNA but after the publication of this new article, I want to try again using some modifications and see if I can get it to work with less RNA. So I can´t really share it right now, sorry!
Best,
Simone

Hi Simone,
Sounds really great that you do have a protocol. What I wonder is RT condition for non-fragmented RNA. Just incubate the RNA with TGIRT at proper temperature for 15 min? Looking forward to your successful modified protocol.

Gary

[chaichontat]

Hi Simone,

I'm trying to amplify one gene of interest; however, I couldn't get a specific band when I added the whole 10 ul of the RT reaction (final PCR volume was 25 ul). The band only appeared when I added 1 ul of the RT mix, suggesting that there's some inhibition of PCR by the RT mix.

However, there was one deviation from your protocol: I used the component version (not ReadyMix) of the KAPA HiFi Hot Start. Do you think that this could have been a factor?

Thanks!
Chaichontat

[kobeho24]

SSII contamination

Hi all,
Recently I’ve done 3 runs of single-cell RNA-seq (Smart seq2) trial. I just followed the original protocol. A highly reproducible background curve can be observed even in negative controls (only nuclease free water) when using our new SuperScript II RTase, which cannot be seen when using SSIII, or without RTase. Our bad SSII lot no. is 1688365.






Does any of you guys have any recommendation of substitutive MMLV RTase? I saw ProtoScripts II & SS IV so far, any candidate else?

Best wishes!
Gary

[Simone78]

Hi all,
Recently I’ve done 3 runs of single-cell RNA-seq (Smart seq2) trial. I just followed the original protocol. A highly reproducible background curve can be observed even in negative controls (only nuclease free water) when using our new SuperScript II RTase, which cannot be seen when using SSIII, or without RTase. Our bad SSII lot no. is 1688365.
[ATTACH]4169[/ATTACH]

[ATTACH]4170[/ATTACH]

[ATTACH]4171[/ATTACH]

Does any of you guys have any recommendation of substitutive MMLV RTase? I saw ProtoScripts II & SS IV so far, any candidate else?

Best wishes!
Gary

I would use either SMARTscribe (Clontech) or Maxima H- (Thermo Scientific).

[kobeho24]

I would use either SMARTscribe (Clontech) or Maxima H- (Thermo Scientific).

Hi Simone,
Between these two, which one's better? It seems that SMARTscribe is similar to SSII and Maxima H- is similar to SSIV. Besides, as SMARTscribe is 100U/ul while Maxima H- is 200U/ul, do we still make it 100U in the 10ul RT reaction as it is in the Smart-seq2 protocol? Am I right?

Best!
Gary