RNA-seq from single cells?

[Timer123]

I am a student from Huazhong Agricultural University，Wuhan，China.Now I am working with the single cell RNA-seq library preparation,because we have so many sample to operate, we could not afford this by use standard Nextera kit,so we have to produce in-house Tn5 transposase by reference the article of "Tn5 transposase and tagmentation procedures for massively scaled sequencing projects"Picelli et al.Genome Research.2014.
In the assemble protocol,where I can get a most suitable the phosphorylation primer:
Tn5MErev, 5'-[phos]CTGTCTCTTATACACATCT-3',and is there any detail protocol or notes about Tn5 transposase assemble and purification ?
And for the smart-seq2, how to order the TSO primer also a problem in China, I know the Exiqon have the patent for the LNA,and order is very difficult. So how to order it faster or is there any good alternative company I can get this?
Thank you for your generous help!

[Simone78]

Originally Posted by Timer123

I am a student from Huazhong Agricultural University，Wuhan，China.Now I am working with the single cell RNA-seq library preparation,because we have so many sample to operate, we could not afford this by use standard Nextera kit,so we have to produce in-house Tn5 transposase by reference the article of "Tn5 transposase and tagmentation procedures for massively scaled sequencing projects"Picelli et al.Genome Research.2014.
In the assemble protocol,where I can get a most suitable the phosphorylation primer:
Tn5MErev, 5'-[phos]CTGTCTCTTATACACATCT-3',and is there any detail protocol or notes about Tn5 transposase assemble and purification ?
And for the smart-seq2, how to order the TSO primer also a problem in China, I know the Exiqon have the patent for the LNA,and order is very difficult. So how to order it faster or is there any good alternative company I can get this?
Thank you for your generous help!

I replied you in a private message, take a look there!

[Timer123]

I have read that，it's very useful for me, thank you very much!

[rnaseqmonkey]

Did anyone solve the mystery of bacterial rRNA contaminations yet?
We're also seeing this pop up inversely correlated to the endogenous RNA amount. We use Maxima H- RT for our libraries, too!

[RevTK]

Dear all,
I`m working with laser-capture microdissected cells (e.g. isolated a few hundred cell equivalents from frozen tissue sections) from mouse tissue. I`m using the single-cell lysis buffer from clontech to generate a lysate of my captured cells right after LCM and then use that lysate directly for the cDNA generation. According to this publication, SMART-Seq2 from this lysate should provide decent results: https://www.ncbi.nlm.nih.gov/pubmed/27387371

After 21 preamplification cycles I get the cDNA profiles seen in the attached .pdf.
The kit used is the SMART-Seq2 Ultra-low v3.

Instead of the expected peak at 1.5-2kb, I get a wide range of smaller peaks across the whole range.
Does this mean degraded RNA? Or possibly fragmented due to the UV laser?
Do these profiles look like too many preamp-cycles were used?
In the above mentioned publication, they also seem to get somewhat of a shift towards smaller cDNA fragments when directly lysing 50 to 120 cells.
But they still got good sequencing results.

If my RNA is fragmented, oligo-dT priming / the used kit won`t work properly I assume.

Is there any chance for a successful sequencing from these kinds of samples? Would switching to a totalRNA-kit that uses random priming improve my chances?

Thank you for your help.

[lmw.bio]

Hi RevTK, wondering if you found a way to sequence your small cDNA samples? Or if you figured out why you got the shorter reads in the first place?

[RevTK]

We tried to make another library with the mammalian pico kit, but the single-stranded DNA in my samples served as template for the RT used in that kit and we got mostly amplification of that.
We just went with the Smart-Seq2 anyways and we got sequencing results. I got about 9 Million reads per sample, out of which between 3 to 6 Million reads were mappable.
Thus I had the libraries sequenced on a second lane, to increase the amount of reads I got per sample.
Now I've around 9-11 Million reads for each sample that were mapped successfully and my results show that I was successful in capturing my target tissue.

There's just some difficulties with a lot of targets that have very low amounts of reads (< 10 reads), of course for these you get gigantic fold-changes if you conduct differential expression analysis using e.g. DESeq2.
I got quite a bit of PolyT contamination in my reads, explaining why a decent amount of the raw reads ended up being unmappable.

I think the data I generated in this way is not of the highest quality, but it can be used for its intended purpose.

[shilpij01]

Hey,
I was just wondering if there is any way to make the rRNA depletion solution on our own?
Thanks
SJ

[nucacidhunter]

Originally Posted by shilpij01

Hey,
I was just wondering if there is any way to make the rRNA depletion solution on our own?
Thanks
SJ

Look at this RNAseH based method: http://journals.plos.org/plosone/art...one.0042882#s4

[Simone78]

Originally Posted by shilpij01

Hey,
I was just wondering if there is any way to make the rRNA depletion solution on our own?
Thanks
SJ

Hi,
there are multiple options. It mostly depends on which method you want to use for depletion: RNAse H, DSN method post-amplification, bead capture. HEre´s a quick summary but let me know if you need more details:
- US Pat 9,428,794: Ribogone from Takara/Clontech
- US Pat 20150299771: Morlan and Sincropi oligos, SDRNA method (see also the 2012 PLoS One paper mentioned below)
- modified SDRNA from Adiconis et al. (Nature Methods 2013, sequences in the Supplementary)
- Probe-directed Degradation, PDD as in Archer et al. (BMC Genomics 2014). They used DSN (Duplex Specific Nuclease), an enzyme also present in Illumina library prep kits and used for normalization. Plenty of papers on the subject are available.
- US Pat 20090137415: method from Euclid Diagnostics (company doesn´t exist anymore, as far as I know)
- EU Patent WO 2011/019993: method used by Epicentre
- US Pat 20160362680: method used by Nugen Technologies (AnyDeplete kit)
- EU Pat WO 2014044724: method used by Qiagen (GeneRead rRNA depletion kit)

Have fun!
/Simone

[skannan4]

Sorry if this has been addressed before on this thread or elsewhere, but for those using ERCC spike-ins, how stable are they? Say, for example, we are using 1:100,000 dilution - how stable is this at -80C?

[Simone78]

Originally Posted by skannan4

Sorry if this has been addressed before on this thread or elsewhere, but for those using ERCC spike-ins, how stable are they? Say, for example, we are using 1:100,000 dilution - how stable is this at -80C?

it is not recommended to freeze and thaw them more than 4 times, according to Ambion. Upon arrival we generate hundreds of tubes with 15 ul of our final dilution (1:4M or 1:40M). 15 ul is the volume necessary for a 384-w plate in our settings, so every tube is enough for 1 plate or 10 plates if using the lower dilution. In this way we can take a single vial when we need it and throw away any leftover.
However, even this is apparently not enough to make experiments very comparable, according to a recent paper where they saw that every freeze and thaw cycle decreases the amount of ERCC by 20%: https://www.ncbi.nlm.nih.gov/pubmed/28263961

...just the last of the many issues of using ERCC!

Best,
Simone

[skannan4]

Thanks for the tips! We'll have to try your approach to avoid the freeze thaw. Do you have a recommended approach for titrating the ERCC before sequencing (for example, is there some characteristic readout on Bioanalyzer)?

[mike137]

Originally Posted by SunPenguin

Has anyone here looked at what the TSO would be doing in subsequent PCR reactions (i.e. could it cause TSO founded amplification)?

I remember reading a protocol a few years back where they inserted several dU in the TSO sequence, then came in with USER enzyme following RT to digest the TSO (both free and incorporated). Does anyone know what that paper was? I'd like to try that.

Bead purification will also remove the TSO, but like many in this thread, I'm not crazy about post-RT purification. Need every single molecule for my PCR!

[Timer123]

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

[Simone78]

Originally Posted by Timer123

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/c56dbb6...204c0cota5.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]

Originally Posted by 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

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]

Originally Posted by cmbetts

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.