I curious to know, (since Linnarsson is in your neighbourhood) have you tested your TSO with UMI for smart2-seq.
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
-
Hi Simone,
Thanks very much for all of your comments and recommendations in this thread.
I am working to test/optimize the Smart-seq2 protocol for a Tcell application and am wondering how you blocked your TSOs. You mentioned iso-nucleotides earlier on in this thread. Is this what you were referring to?
iso-dG: http://www.idtdna.com/site/catalog/m...s/product/2188
iso-dC: http://www.idtdna.com/site/catalog/m...s/product/2187
description: http://www.idtdna.com/Site/Catalog/M...ons/Category/7
I have little experience with such nucleotides in the past. Is the idea that iso-dG and 5Me-iso-dC pair with each other and not with regular A,C,G,T, thus the RT stops when it comes across an iso-dC or -dG because its counterpart does not exist in the dNTP mix? Is concatemerization prevented then by obstructing the RT before it can add another round of untemplated Cs after the copying the TSO?
Best,
DanLast edited by dtm2451; 07-30-2014, 09:30 AM.
Comment
-
There is an extended discussion about oligo blocking on previous pages in this thread. Other groups are using 5' biotin as a cheaper way of doing the same thing, but it sounds like you understand the principle. See doi:10.1186/1471-2164-11-413 for more.
Comment
-
Thank you jwfoley. I am actually thinking of trying both and testing biotin vs. iso vs. unblocked. But, I'm not sure how to get an oligo with both iso and LNA. Looking back, you actually mentioned this issue before. Were you able to find a place to order an LNA-iso combo or did you switch to biotin and avoid the issue? If not, has anyone else been able to order an oligo containing both locked and iso nucleotides? And if so, where from?
Comment
-
No, I never tried iso because biotin completely eliminated the hedgehog in my tests. However, if you talk to Exiqon's sales reps, they seem flexible about ordering things that are "off the menu" (since they apparently just subcontract their orders out to companies like IDT anyway).
Comment
-
Originally posted by dtm2451 View PostThank you jwfoley. I am actually thinking of trying both and testing biotin vs. iso vs. unblocked. But, I'm not sure how to get an oligo with both iso and LNA. Looking back, you actually mentioned this issue before. Were you able to find a place to order an LNA-iso combo or did you switch to biotin and avoid the issue? If not, has anyone else been able to order an oligo containing both locked and iso nucleotides? And if so, where from?
Comment
-
Originally posted by MMaria View PostInteresting. Does anybody knows how much does it cost or which labs already have it?
Comment
-
Hi seqgirl,
I use a rough estimation from the bioanalyzer (the Qbit minimum RNA amount is too high for such low quantities); if you have single cells RNA, I don't think there is any way to quantify it (or even get the RNA tracks w/ the bioanalyzer).
Guy
Comment
-
If these kits are being used for low input, it is a good idea to QC input RNA because kits are expensive. When using single-cells in C1 it is physically impossible to QC RNA and the approach is to check success after harvesting amplifid cDNA. The same goes if single-cells are processed manually.
Comment
-
Does everyone here do cell-sorting and cDNA synthesis on the same day? has anyone here tried to use pre-frozen sorted single cell with SMARTSeq protocol?
We tried but only a very few of reactions worked so far. Wanted to know where we can troubleshooting. Really appreciate your input.
Thanks
Comment
-
1-5ng input
Originally posted by CTC View PostThanks for the article. But without detailed protocol it is hard to replicate the experiment.
I have tried SMARTer PCR kit but it did not work in my hands. The expected yield for a single cell is 7 ng, the negative control yield is similar.
The procedure takes 5 hours - librairies are oriented.
It works on Eurcaryotes. There are actually 3 methods depending on the goal of the project. One is based only OligodT and you would see only 5% of reads with rRNA.
Comment
-
Originally posted by ChristmasSunflower View PostDoes everyone here do cell-sorting and cDNA synthesis on the same day? has anyone here tried to use pre-frozen sorted single cell with SMARTSeq protocol?
We tried but only a very few of reactions worked so far. Wanted to know where we can troubleshooting. Really appreciate your input.
Thanks
Comment
-
bioanalyzer not correlating with seq data?
Hello all, thanks for all your thoughts here. It's helpful.
Just curious about how well your bioanalyzer traces correlate with actual sequencing data. With our samples, we can't see much of a correlation.
For example, in the attached pdf, sample D2 appears to have some concatamer and that sample mapped poorly to mouse genome (8%) and we detected very few genes (less than 200). Sample C8 seems to have a higher amount of product (between 500 and 10,000 bp) and concatemer. But this sample mapped 50% to mouse genome, and we we detected almost 5000 genes. Sample G5 has no concatamer, primer dimer (at ~100 bp), and noticeable product, but had 14% mapping and 600 genes detected.
In short, we can’t seem to find a correlation between what we observe on the bioanalyzer trace and what we’re seeing with sequencing, at least by mapping % and genes detected. We’re actually considering skipping bioanalyzer since sequencing costs on a miSeq for 96 samples (~$1000) is about equal to two Bioanalyzer chips (11 samples per chip) anyway. Anyone have any thoughts on this?
Thanks.
Comment
Latest Articles
Collapse
-
by seqadmin
Next-generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR) are essential techniques for investigating the genome, transcriptome, and epigenome. In many cases, choosing the appropriate technique is straightforward, but in others, it can be more challenging to determine the most effective option. A simple distinction is that smaller, more focused projects are typically better suited for qPCR, while larger, more complex datasets benefit from NGS. However,...-
Channel: Articles
10-18-2024, 07:11 AM -
-
by seqadmin
Non-coding RNAs (ncRNAs) do not code for proteins but play important roles in numerous cellular processes including gene silencing, developmental pathways, and more. There are numerous types including microRNA (miRNA), long ncRNA (lncRNA), circular RNA (circRNA), and more. In this article, we discuss innovative ncRNA research and explore recent technological advancements that improve the study of ncRNAs.
Nobel Prize for MicroRNA Discovery
This week,...-
Channel: Articles
10-07-2024, 08:07 AM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
New Model Aims to Explain Polygenic Diseases by Connecting Genomic Mutations and Regulatory Networks
by seqadmin
Started by seqadmin, Yesterday, 05:31 AM
|
0 responses
10 views
0 likes
|
Last Post
by seqadmin
Yesterday, 05:31 AM
|
||
Started by seqadmin, 10-24-2024, 06:58 AM
|
0 responses
20 views
0 likes
|
Last Post
by seqadmin
10-24-2024, 06:58 AM
|
||
New AI Model Designs Synthetic DNA Switches for Targeted Gene Expression in Specific Cell Types
by seqadmin
Started by seqadmin, 10-23-2024, 08:43 AM
|
0 responses
48 views
0 likes
|
Last Post
by seqadmin
10-23-2024, 08:43 AM
|
||
Started by seqadmin, 10-17-2024, 07:29 AM
|
0 responses
58 views
0 likes
|
Last Post
by seqadmin
10-17-2024, 07:29 AM
|
Comment