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Old 12-27-2016, 05:46 PM   #241
Timer123
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Location: Wuhan.China

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Default Tn5 assemble and single cell sequence

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!
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Old 12-30-2016, 01:06 AM   #242
Simone78
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Default

Quote:
Originally Posted by Timer123 View Post
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!
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Old 01-01-2017, 11:03 PM   #243
Timer123
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Default Tn5 assemble and single cell sequence

I have read that,it's very useful for me, thank you very much!
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Old 01-18-2017, 10:45 PM   #244
rnaseqmonkey
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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!
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Old 04-02-2017, 11:20 PM   #245
RevTK
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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.
Attached Files
File Type: pdf 21preamp_samples.pdf (211.5 KB, 37 views)

Last edited by RevTK; 04-02-2017 at 11:22 PM.
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Old 08-16-2017, 09:14 AM   #246
lmw.bio
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Arrow Sequencing small cDNA libraries

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?
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Old 08-16-2017, 10:54 PM   #247
RevTK
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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.
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