[Simone78]

Dear all:
Recently, I have done a few cell（About 500-1000cell） RNA-seq by smart-seq2 protocol. After the RT and PCR amplification reaction, a bright band stuck in the well after agarose gel electrophoresis. I used KAPA HiFi HotStart Readymix for my PCR reaction(All regents follow smart-seq2). Dose anyone meet the same problem in smart-seq2? I hope I can get some feedback on what caused this. I have attached a picture for your reference.Thank you very much!
https://www.researchgate.net/profile...%E7%89%871.png

Hi,
I would say the reaction failed completely and what you see still in the well is gDNA. I would first try SS2 on some good quality RNA (10 pg, 100 pg, 1 ng or higher), to eliminate issues with cell-to-cell variability and cell quality. Moreover, sorting 500-1000 cells in 0.2% Triton might lead to incomplete lysis. I would always use the Bioanalyzer/Tapestation to check the cDNA quality as the agarose gel is not sensitive enough. If you need some more help, just let me know!
Best,
Simone

[Timer123]

Dear professor Simone:
I am very grateful for your reply so quickly. Thank you. Your advice will be a great way to check our reaction successful or not. And I will try it recently.
Before that I omitted an important message: our material is about a few hundred plant cells.
Previously, I got a good quality RNA-seq library (Ensure by data analysis), which also check the preamplification reaction by agarose gel electrophoresis, and as the picture shows, with bright bands stuck in the well. We still do not kown why this happens?

https://wx3.sinaimg.cn/mw690/c56dbb6dgy1fr2vm4r3jtj204c0cota5.jpg

And this time, I follow the protocol again(with the same material), a very lower ds cDNA field (4 times less) I get, it really make me crazy, do you have any suggestion?
Best
Student YangKe

[ChristmasSunflower]

Hello,

I recently used Smart_Seq2 protocol for single cell study. These single cell is manually picked up into 8-strip PCR tube. I designed all 3 primers TSO, Oligo-dT30VN, and ISPCR oligo with biotin modification in 5' side. I did a few of experimental samples, along with RNA positive control RNA 1ng, 100pg and 10pg, and water negative control. I did 18 PCR cycles for preamplification and 1:1 beads ratio for PCR cleanup. From Qubit reading, it's hard to say if protocol worked. I run them with Bioanalyzer. It seems results are positive. Can someone here with more experimental experience comment this result?
https://www.dropbox.com/s/htc0pp0f8r...Afile.jpg?dl=0

Thanks

[cmbetts]

Hello,

I recently used Smart_Seq2 protocol for single cell study. These single cell is manually picked up into 8-strip PCR tube. I designed all 3 primers TSO, Oligo-dT30VN, and ISPCR oligo with biotin modification in 5' side. I did a few of experimental samples, along with RNA positive control RNA 1ng, 100pg and 10pg, and water negative control. I did 18 PCR cycles for preamplification and 1:1 beads ratio for PCR cleanup. From Qubit reading, it's hard to say if protocol worked. I run them with Bioanalyzer. It seems results are positive. Can someone here with more experimental experience comment this result?
https://www.dropbox.com/s/htc0pp0f8r...Afile.jpg?dl=0

Thanks

There's tons of low MW products there indicating massive RNA degradation, which you have in both your cell and purified RNA samples. Did you check the RIN of your pure RNA? If it's high, then you've got an RNase contamination problem, otherwise you need to look at your cell/RNA isolation procedure. I would not proceed to library prep and sequencing with those samples.

[Simone78]

There's tons of low MW products there indicating massive RNA degradation, which you have in both your cell and purified RNA samples. Did you check the RIN of your pure RNA? If it's high, then you've got an RNase contamination problem, otherwise you need to look at your cell/RNA isolation procedure. I would not proceed to library prep and sequencing with those samples.

I agree, the RNA controls should be much better and give tons of cDNA, especially the 100 pg and 1 ng. Lots of degraded stuff or leftover primers in general. S2 and S7 are decent, though. S3 and S4 still have cDNA left, the others are all degraded. You might want to be more stringent with bead cleanup (0.8X is sufficient) and/or decrease the amount of oligos you are using. Which cells are these, by the way? That would determine the number of PCR cycles and help understanding whether it´s possible to get some good cDNA in general. Although Smart-seq2 is rather robust, not all cells give good results and you might need to play around with the lysis buffer.

[ChristmasSunflower]

There's tons of low MW products there indicating massive RNA degradation, which you have in both your cell and purified RNA samples. Did you check the RIN of your pure RNA? If it's high, then you've got an RNase contamination problem, otherwise you need to look at your cell/RNA isolation procedure. I would not proceed to library prep and sequencing with those samples.

Yes, RNA control quality is checked by Bioanalyzer with the perfect RIN=10. I did parallel 2 technical replicates with RNA 1ng, RNA100pg and RNA10pg and just didn't get chance to load to this 11-well HS chip. But if you looked at my negative control, there is also big peak in the left size so I initially thought it's some other things.

Thanks

[ChristmasSunflower]

Yes, RNA control quality is checked by Bioanalyzer with the perfect RIN=10. I did parallel 2 technical replicates with RNA 1ng, RNA100pg and RNA10pg and just didn't get chance to load to this 11-well HS chip. But if you looked at my negative control, there is also big peak in the left size so I initially thought it's some other things.

Thanks

[ChristmasSunflower]

I agree, the RNA controls should be much better and give tons of cDNA, especially the 100 pg and 1 ng. Lots of degraded stuff or leftover primers in general. S2 and S7 are decent, though. S3 and S4 still have cDNA left, the others are all degraded. You might want to be more stringent with bead cleanup (0.8X is sufficient) and/or decrease the amount of oligos you are using. Which cells are these, by the way? That would determine the number of PCR cycles and help understanding whether it´s possible to get some good cDNA in general. Although Smart-seq2 is rather robust, not all cells give good results and you might need to play around with the lysis buffer.

They are mouse sensory neuron cells. I wonder why there are same things in the left side in my negative controls. I thought I got some cDNA amplifications in experimental samples. Yes, these pure RNA control results are puzzled me.

Thanks

[Simone78]

They are mouse sensory neuron cells. I wonder why there are same things in the left side in my negative controls. I thought I got some cDNA amplifications in experimental samples. Yes, these pure RNA control results are puzzled me.

Thanks

the peak on the left, in my opinion, is primers/dimers. Unused primers, either because there is no RNA (NC) or degraded RNA will be amplified and accumulate in the reaction. If the RNA is little or degraded, the amount of primers is in large excess (it´s in large excess anyway, but this is another story...). Maybe the amount is so high that the bead cleanup can´t remove them entirely. Cutoff of beads is around 100 bp but even shorter fragments are retained.

[ChristmasSunflower]

the peak on the left, in my opinion, is primers/dimers. Unused primers, either because there is no RNA (NC) or degraded RNA will be amplified and accumulate in the reaction. If the RNA is little or degraded, the amount of primers is in large excess (it´s in large excess anyway, but this is another story...). Maybe the amount is so high that the bead cleanup can´t remove them entirely. Cutoff of beads is around 100 bp but even shorter fragments are retained.

Hi Simone,

I thought they are some primer leftovers after beads cleanup. But I do see the amplification of cDNA in most experimental samples, such as S#1, , 3, 4 5 and 7, right? There are consistent peak around 2Kb in most of experimental samples, I thought it's a good sign.

Thanks

[sbarberan]

Talking about which cells are used, is there anyone here with experience with single-cell experiments with MCF7?

Also, Simeone78, when you say play around with the lysis buffer, what exactly do you play around with?

[ChristmasSunflower]

Talking about which cells are used, is there anyone here with experience with single-cell experiments with MCF7?

Also, Simeone78, when you say play around with the lysis buffer, what exactly do you play around with?

Yes, I want to know how to play with lysis buffer?

Thanks

[Simone78]

Yes, I want to know how to play with lysis buffer?

Thanks

One could try to increase the amount of Triton or use other (non-ionic) detergents such as Tween-20, Igepal CA-630, NP-40 or BSA (as in Svec et al. 2013, PMID: 24224157). Maybe some other RNAse inhibitor would help. In my opinion, SuperaseIn (Thermo Fisher) is better than Rec RNAse inh. from Takara used for the Smart-seq2 protocol. We just don´t use SuperaseIn because of the higher cost.
If you use the RNAse inh. from Takara you might want to add some DTT (as in the STRT-seq and STRT-seq 2i papers from S. Linnarsson), since the RNAse inh. requires DTT for working in an optimal way.
Definitely don´t use SDS, as ionic detergents kill the enzymes, no matter which concentration you do the lysis.
In a recent paper I have also seen RLT buffer (5M guanidine isothiocyanate) but I can´t recall which one, sorry. It might be some kind of modified scATAC-seq paper.
There are also many patents exploring the subject. I am especially fond of US 2011/0136180A1, for example

Best,
Simone

[ChristmasSunflower]

Thanks, Simone! You have a big play in this discussion topic and I believe many people have benefited from this discussion. Really appreciate!


Thanks

[thang198]

we have recently published a paper where we describe Smart-seq2, an improvement of the Smarter kit. Check it out! You don´need any kit to make your cDNA and you´re going to save a lot of money!

http://www.ncbi.nlm.nih.gov/pubmed/24385147

http://www.ncbi.nlm.nih.gov/pubmed/24056875

Hi Simone78,

Would you please let me know that I can modify your Nature protocol paper "Full-length RNA-seq from single cells using Smart-seq2" for the bulk RNA samples?
I do have 60 different RNA samples but the Truseq from Illumina is expensive. I found that your protocol may reduce the cost since the Nextera is cheaper.
Thank you,